Metastasis-associated antigen and antibodies thereto

ABSTRACT

There are provided membrane-associated polypeptides having the sequence shown in SEQ ID Nos. 1 and 9. Also provided are immunogenic determinants derived from said polypeptides and antibodies raised thereto. The polypeptides, their derived antigenic determinants and the antibodies are useful for the diagnosis and treatment of metastatic potential in tumors.

This application is the National Stage filing under 35 U.S.C. 371 of PCT International Application ser. No. PCT/EP97/01963 (designating the United States), filed Apr. 18, 1997, which claims priority under 35 U.S.C. 119(e) from U.S. Ser. No. 60/016,487, filed Apr. 29, 1996 now abandoned.

The present invention relates to a novel antigen which is closely associated with metastatic behaviour of tumour cells in vitro and in vivo, as well as to antibodies recognising said antigen and uses thereof in diagnosis and treatment of malignancies.

Metastasis is a multi-stage process by which tumour cells leave the site of a primary tumour, enter blood and lymph vessels, migrate to distant parts of the body and form novel foci of tumour growth. In keeping with the apparently complex nature of metastasis, experimental data suggest that no one factor is exclusively responsible for the determination of metastatic potential. Rather, a cascade of events, each of which incrementally increases the liability of a tumour to metastasis, leads to metastasis and tumour spread. Each of the events of the metastatic cascade is not believed to play a role of identical importance in each metastatic situation. A given metastatic cell is likely to possess some less developed metastatic properties, but still possess high metastatic potential because other properties are highly pronounced.

For this reason, a reliable marker for metastatic potential is difficult to determine. There are indications, however, that the cell surface is closely involved with many aspects of the metastatic cascade. The detachment of tumour cells, migration through tissues and final attachment to form a new focus of growth are events which all involve the behaviour of the cell surface.

It is known to attempt to raise antibodies directed to antigens preferentially expressed by tumour cells which show metastatic potential. For example, Vollmers and Birchmeier, (1983) PNAS 80, 3739-3733 & 6863-6867, demonstrated that certain antibodies raised to mouse melanoma cell line surface protein were able to inhibit cell adhesion and block metastasis. The antibodies appeared to target antigens of between 40 and 50 kDa.

By raising antisera to membrane associated proteins and assessing cross-reactivity with transformed cells, we have now been able to identify novel 150 kDa cell-surface proteins which correlate extremely closely with metastatic potential both in vitro and in vivo.

SUMMARY OF THE INVENTION

According to the present invention, we provide membrane-associated polypeptides having the sequence shown in SEQ ID NOS. 2 and 9, respectively. Also provided are immunogenic determinants derived from said polypeptides and antibodies raised thereto. The polypeptides, their derived antigenic determinants and the antibodies are useful for the diagnosis and treatment of metastatic potential in tumours.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the deduced amino acid sequence of the metastasis-related antigen p150 isolated from mouse. The sequence data derived by amino acid microsequencing is underlined.

FIG. 2 shows a sequence homology comparison between p150 from mouse and related sequences.

DETAILED DESCRIPTION OF THE INVENTION

By raising monoclonal antibodies to membrane fractions derived from human placenta and subsequently screening the antibodies for cell surface staining in transformed versus non-transformed cells, an antibody which identifies an antigen preferentially expressed on transformed cells can be identified. This antigen, referred to herein as p150 from mouse, has an apparent molecular weight on SDS-PAGE of ca. 150 kDa and the sequence shown in SEQ ID No. 2. The human homologue is represented in SEQ ID No. 9.

The antigens are expressed in tumours, tumour cell lines and primary cells derived from tumours, and appear at much lower levels in normal human tissue. The levels of expression of the antigens are not consistent between tumours and cell lines derived therefrom, including primary cell lines. However, within each separate system, we have determined that the antigens closely correlate with metastatic potential. The level of expression of the antigens appears to be dependent on the differentiation state of the tumour cell. Thus, relatively undifferentiated tumour cells, which have a high metastatic potential, express relatively high levels of p150, while more differentiated cells express a lower amount of these antigens. Tumour tissue derived from actual metastases appears to express the most elevated levels of p150.

The present invention includes the p150 proteins both from mouse and human and all derivatives thereof which are useful for diagnosing the metastatic potential of a tumour. Preferably, unless otherwise indicated or implied, such derivatives are encompassed by the term “p150”. The high degree of correlation of p150 with metastatic potential makes the antigens useful for determining the correct treatment to be used, as it is known that a high metastatic potential correlates with a poor prognosis. Accordingly, the derivatives of p150 comprised in the invention include all those which retain characteristics of p150 which allow them to be identified in diagnostic assays. The preferred characteristic retained by the p150 derivatives of the invention is a common antigenic determinant shared with p150.

“Common antigenic determinant” means that the derivative in question at least one antigenic function of p150. Antigenic functions includes possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring or denatured p150 polypeptide or fragment thereof. Thus p150 as provided by the present invention includes splice variants encoded by mRNA generated by alternative splicing of a primary transcript, amino acid mutants, glycosylation variants and other covalent derivatives of p150 which retain the physiological and/or physical properties of p150. Exemplary derivatives include molecules wherein the protein of the invention is covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid. Such a moiety may be a detectable moiety such as an enzyme or a radioisotope. Further included are naturally occurring variants of p150 found with a particular species, preferably a mammal. Such a variant may be encoded by a related gene of the same gene family, by an allelic variant of a particular gene, or represent an alternative splicing variant of the p150 gene.

Derivatives which retain common antigenic determinants can be fragments of p150. Fragments of p150 comprise individual domains thereof, as well as smaller polypeptides derived from the domains. Preferably, smaller polypeptides derived from p150 according to the invention define a single epitope which is characteristic of p150. Fragments may in theory be almost any size, as long as they retain one characteristic of p150. Preferably, fragments will be between 5 and 500 amino acids in length. Longer fragments are regarded as truncations of the full-length p150 and generally encompassed by the term “p150”.

Derivatives of p150 also comprise mutants thereof, which may contain amino acid deletions, additions or substitutions, subject to the requirement to maintain at least one feature characteristic of p150. Thus, conservative amino acid substitutions may be made substantially without altering the nature of p150, as may truncations from the 5′ or 3′ ends. Deletions and substitutions may moreover be made to the fragments of p150 comprised by the invention. p150 mutants may be produced from a DNA encoding p150 which has been subjected to in vitro mutagenesis resulting e.g. in an addition, exchange and/or deletion of one or more amino acids. For example, substitutional, deletional or insertional variants of p150 can be prepared by recombinant methods and screened for immuno-crossreactivity with the native forms of p150.

The fragments, mutants and other derivative of p150 preferably retain substantial homology with p150. As used herein, “homology” means that the two entities share sufficient characteristics for the skilled person to determine that they are similar in origin and function. Preferably, homology is used to refer to sequence identity. Thus, the derivatives of p150 preferably retain substantial sequence identity with the sequences of SEQ ID Nos. 2 and 9, respectively.

“Substantial homology”, where homology indicates sequence identity, means more than 50% sequence identity, preferably more than 75% sequence identity and most preferably a sequence identity of 90% or more.

Preferably, the proteins or derivatives thereof of the invention are provided in isolated form. “Isolated” means that the respective protein or derivative thereof has been identified and is free of one or more components of its natural environment. Isolated p150 includes p150 in a recombinant cell culture. P150 present in an organism expressing a recombinant p150 gene, whether the p150 protein is “isolated” or otherwise, is included within the scope of the present invention.

The polypeptides according to the invention are closely associated with the metastatic process in tumour cells. Accordingly, the invention provides a composition comprising a polypeptide according to the invention or an antagonist thereto for use as a medicament in the treatment or diagnosis of tumours.

According to a further aspect of the present invention, there are provided nucleic acids encoding p150. In addition to being useful for the production of recombinant p150 protein, these nucleic acids are also useful as probes, thus readily enabling those skilled in the art to identify and/or isolate nucleic acid encoding p150. The nucleic acid may be unlabelled or labelled with a detectable moiety. Furthermore, nucleic acids according to the invention are useful e.g. in a method determining the presence of p150-specific nucleic acid, said method comprising hybridising the DNA (or RNA) encoding (or complementary to) p150 to test sample nucleic acid and determining the presence of p150. In another aspect, the invention provides nucleic acid sequences that are complementary to, or hybridise under stringent conditions to, a nucleic acid sequence encoding p150.

The invention also provides a method for amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase (chain) reaction with nucleic acid (DNA or RNA) encoding (or complementary to) p150.

In still another aspect of the invention, the nucleic acid is DNA and further comprises a replicable vector comprising the nucleic acid encoding p150 operably linked to control sequences recognised by a host transformed by the vector. Furthermore the invention provides host cells transformed with such a vector and a method of using a nucleic acid encoding p150 to effect the production of p150, comprising expressing p150 nucleic acid in a culture of the transformed host cells and, if desired, recovering p150 from the host cell culture.

Furthermore, the present invention relates to isolated p150 proteins and derivatives thereof encoded by the above-described nucleic acids.

Isolated p150 nucleic acid includes a nucleic acid that is free from at least one contaminant nucleic acid with which it is ordinarily associated in the natural source of p150 nucleic acid or in crude nucleic acid preparations, such as DNA libraries and the like. Isolated nucleic acid thus is present in other than in the form or setting in which it is found in nature. However, isolated p150 encoding nucleic acid includes p150 nucleic acid in ordinarily p150-expressing cells where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different DNA sequence than that found in nature.

In accordance with the present invention, there are provided isolated nucleic acids, e.g. DNAs or RNAs, encoding p150, particularly mammalian p150, e.g. human p150, or fragments thereof. In particular, the invention provides DNA molecules encoding p150, or a fragment thereof. By definition, such a DNA comprises a coding single stranded DNA, a double stranded DNA of said coding DNA and complementary DNA thereto, or this complementary (single stranded) DNA itself. Exemplary nucleic acids encoding p150 are represented in SEQ ID Nos. 1 and 8.

The preferred sequences encoding p150 are those having substantially the same nucleotide sequence as the coding sequences in SEQ ID Nos. 1 and 8, with the nucleic acids having the same sequence as the coding sequences in SEQ ID Nos. 1 and 8 being most preferred. As used herein, nucleotide sequences which are substantially the same share at least about 90% identity. However, in the case of splice variants having e.g. an additional exon sequence homology may be lower.

The nucleic acids of the invention, whether used as probes or otherwise, are preferably substantially homologous to the sequences of p150 as shown in SEQ ID Nos. 1 and 8. The terms “substantially” and “homologous” are used as hereinbefore defined with reference to the p150 polypeptide(s).

Preferably, nucleic acids according to the invention are fragments of the p150-encoding sequences, or derivatives thereof as hereinbefore defined in relation to polypeptides. Fragments of the nucleic acid sequences of a few nucleotides in length, preferably 5 to 150 nucleotides in length, are especially useful as probes.

Exemplary nucleic acids can alternatively be characterised as those nucleotide sequences which encode a p150 protein and hybridise to the DNA sequences set forth SEQ ID Nos. 1 and 8, respectively, or a selected fragment of said DNA sequences. Preferred are such sequences encoding p150 which hybridise under high-stringency conditions to the sequences of SEQ ID Nos. 1 and 8, respectively.

Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skill in the field. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (T_(m)) of the hybrid which decreases approximately 1 to 1.5° C. with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridisation reaction is performed under conditions of higher stringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na⁺ at 65-68° C. High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6×SSC, 5×Denhardt's, 1% SDS (sodium dodecyl sulphate), 0.1 Na⁺ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific competitor. Following hybridisation, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2−0.1×SSC, 0.1% SDS.

Moderate stringency refers to conditions equivalent to hybridisation in the above described solution but at about 60-62° C. In that case the final wash is performed at the hybridisation temperature in 1×SSC, 0.1% SDS.

Low stringency refers to conditions equivalent to hybridisation in the above described solution at about 50-52° C. In that case, the final wash is performed at the hybridisation temperature in 2×SSC, 0.1% SDS.

It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g. formamide-based buffers, and temperatures. Denhardt's solution and SSC are well known to those of skill in the art as are other suitable hybridisation buffers (see, e.g. Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds. (1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). Optimal hybridisation conditions have to be determined empirically, as the length and the GC content of the probe also play a role.

Given the guidance provided herein, the nucleic acids of the invention are obtainable according to methods well known in the art. For example, a DNA of the invention is obtainable by chemical synthesis, using polymerase chain reaction (PCR) or by screening a genomic library or a suitable cDNA library prepared from a source believed to possess p150 and to express it at a detectable level.

Chemical methods for synthesis of a nucleic acid of interest are known in the art and include triester, phosphite, phosphoramidite and H-phosphonate methods, PCR and other autoprimer methods as well as oligonucleotide synthesis on solid supports. These methods may be used if the entire nucleic acid sequence of the nucleic acid is known, or the sequence of the nucleic acid complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, one may infer potential nucleic acid sequences using known and preferred coding residues for each amino acid residue.

An alternative means to isolate the gene encoding p150 is to use PCR technology as described e.g. in section 14 of Sambrook et al., 1989. This method requires the use of oligonucleotide probes that will hybridise to p150 nucleic acid. Strategies for selection of oligonucleotides are described below.

Libraries are screened with probes or analytical tools designed to identify the gene of interest or the protein encoded by it. For cDNA expression libraries suitable means include monoclonal or polyclonal antibodies that recognise and specifically bind to p150; oligonucleotides of about 20 to 80 bases in length that encode known or suspected p150 cDNA from the same or different species; and/or complementary or homologous cDNAs or fragments thereof that encode the same or a hybridising gene. Appropriate probes for screening genomic DNA libraries include, but are not limited to oligonucleotides, cDNAs or fragments thereof that encode the same or hybridising DNA; and/or homologous genomic DNAs or fragments thereof.

A nucleic acid encoding p150 may be isolated by screening suitable cDNA or genomic libraries under suitable hybridisation conditions with a probe, i.e. a nucleic acid disclosed herein including oligonucleotides derivable from the sequences set forth in SEQ ID Nos. 1 and 8. Suitable libraries are commercially available or can be prepared e.g. from cell lines, tissue samples, and the like.

As used herein, a probe is e.g. a single-stranded DNA or RNA that has a sequence of nucleotides that includes between 10 and 50, preferably between 15 and 30 and most preferably at least about 20 contiguous bases that are the same as (or the complement of) an equivalent or greater number of contiguous bases set forth in SEQ ID Nos. 1 and 8, respectively. The nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are minimised. The nucleotide sequences are usually based on conserved or highly homologous nucleotide sequences or regions of p150. The nucleic acids used as probes may be degenerate at one or more positions. The use of degenerate oligonucleotides may be of particular importance where a library is screened from a species in which preferential codon usage in that species is not known.

Preferred regions from which to construct probes include 5′ and/or 3′ coding sequences, sequences predicted to encode ligand binding sites, and the like. For example, either the full-length cDNA clone disclosed herein or fragments thereof can be used as probes. Preferably, nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridisation. For example, a suitable label means is a radiolabel. The preferred method of labelling a DNA fragment is by incorporating a^(32P) dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art. Oligonucleotides are usually end-labelled with g^(32P)-labelled ATP and polynucleotide kinase. However, other methods (e.g. non-radioactive) may also be used to label the fragment or oligonucleotide, including e.g. enzyme labelling, fluorescent labelling with suitable fluorophores and biotinylation.

After screening the library, e.g. with a portion of DNA including substantially the entire p150-encoding sequence or a suitable oligonucleotide based on a portion of said DNA, positive clones are identified by detecting a hybridisation signal; the identified clones are characterised by restriction enzyme mapping and/or DNA sequence analysis, and then examined, e.g. by comparison with the sequences set forth herein, to ascertain whether they include DNA encoding a complete p150 (i.e., if they include translation initiation and termination codons). If the selected clones are incomplete, they may be used to rescreen the same or a different library to obtain overlapping clones. If the library is genomic, then the overlapping clones may include exons and introns. If the library is a cDNA library, then the overlapping clones will include an open reading frame. In both instances, complete clones may be identified by comparison with the DNAs and deduced amino acid sequences provided herein.

In order to detect any abnormality of endogenous p150, genetic screening may be carried out using the nucleotide sequences of the invention as hybridisation probes. Such probes can e.g. be used to screen specific tissues and body fluids like e.g. blood, urine, spinal-fluid, ascites and serum. Also, based on the nucleic acid sequences provided herein antisense-type therapeutic agents may be designed.

It is envisaged that the nucleic acids of the invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof. Such mutants can be used e.g. to produce a p150 mutant that has an amino acid sequence differing from the p150 sequences as found in nature. Mutagenesis may be predetermined (site-specific) or random. A mutation which is not a silent mutation must not place sequences out of reading frames and preferably will not create complementary regions that could hybridise to produce secondary mRNA structure such as loops or hairpins.

The cDNA or genomic DNA encoding native or mutant p150 can be incorporated into vectors for further manipulation. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA amplification or for DNA expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.

Both expression and cloning vectors generally contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically in cloning vectors, these sequences are those which enable the vector to replicate independently of the host chromosomal DNA, and include origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2m plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, polyoma, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors unless these are used in mammalian cells competent for high level DNA replication, such as COS cells.

Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be replicated by insertion into the host genome. However, the recovery of genomic DNA encoding p150 is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise p150 DNA. DNA can be amplified by PCR and be directly transfected into the host cells without any replication component.

Advantageously, an expression and cloning vector may contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.

As to a selective gene marker appropriate for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene. Suitable markers for yeast are, for example, those conferring resistance to antibiotics G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, LYS2, TRP1, or HIS3 gene.

Since the replication of vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from E. coli plasmids, such as pBR322, Bluescript® vector or a pUC plasmid, e.g. pUC18 or pUC19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.

Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up p150 nucleic acid, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes conferring resistance to G418 or hygromycin. The mammalian cell transformants are placed under selection pressure which only those transformants which have taken up and are expressing the marker are uniquely adapted to survive. In the case of a DHFR or glutamine synthase (GS) marker, selection pressure can be imposed by culturing the transformants under conditions in which the pressure is progressively increased, thereby leading to amplification (at its chromosomal integration site) of both the selection gene and the linked DNA that encodes p150. Amplification is the process by which genes in greater demand for the production of a protein critical for growth, together with closely associated genes which may encode a desired protein, are reiterated in tandem within the chromosomes of recombinant cells. Increased quantities of desired protein are usually synthesised from thus amplified DNA.

Expression and cloning vectors usually contain a promoter that is recognised by the host organism and is operably linked to p150 nucleic acid. Such a promoter may be inducible or constitutive. The promoters are operably linked to DNA encoding p150 by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native p150 promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of p150 DNA.

Promoters suitable for use with prokaryotic hosts include, for example, the b-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding p150, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the DNA encoding p150.

Moreover, the p150 genes according to the invention preferably include a secretion sequence in order to facilitate secretion of the corresponding polypeptide from bacterial hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body. The peptide may be recovered from the bacterial periplasmic space, or the culture medium, as appropriate.

Suitable promoting sequences for use with yeast hosts may be regulated or constitutive and are preferably derived from a highly expressed yeast gene, especially a Saccharomyces cerevisiae gene. Thus, the promoter of the TRP1 gene, the ADHI or ADHII gene, the acid phosphatase (PH05) gene, a promoter of the yeast mating pheromone genes coding for the a- or a-factor or a promoter derived from a gene encoding a glycolytic enzyme such as the promoter of the enolase, glyceraldehyde-3-phosphate dehydrogenase (GAP), 3-phospho glycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase or glucokinase genes, or a promoter from the TATA binding protein (TBP) gene can be used. Furthermore, it is possible to use hybrid promoters comprising upstream activation sequences (UAS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene, for example a hybrid promoter including the UAS(s) of the yeast PH05 gene and downstream promoter elements including a functional TATA box of the yeast GAP gene (PH05-GAP hybrid promoter). A suitable constitutive PHO5 promoter is e.g. a shortened acid phosphatase PH05 promoter devoid of the upstream regulatory elements (UAS) such as the PH05 (−173) promoter element starting at nucleotide −173 and ending at nucleotide −9 of the PH05 gene.

p150 gene transcription from vectors in mammalian hosts may be controlled by promoters derived from the genomes of viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or a very strong promoter, e.g. a ribosomal protein promoter, and from the promoter normally associated with p150 sequence, provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding p150 by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are relatively orientation and position independent. Many enhancer sequences are known from mammalian genes (e.g. elastase and globin). However, typically one will employ an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer. The enhancer may be spliced into the vector at a position 5′ or 3′ to p150 DNA, but is preferably located at a site 5′ from the promoter.

Advantageously, a eukaryotic expression vector encoding p150 may comprise a locus control region (LCR). LCRs are capable of directing high-level integration site independent expression of transgenes integrated into host cell chromatin, which is of importance especially where the p150 gene is to be expressed in the context of a permanently-transfected eukaryotic cell line in which chromosomal integration of the vector has occurred, in vectors designed for gene therapy applications or in transgenic animals.

Suitable eukaryotic host cells for expression of p150 include yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms will also contain sequences necessary for the termination of transcription and for stabilising the mRNA. Such sequences are commonly available from the 5′ and 3′ untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNAs encoding p150.

An expression vector includes any vector capable of expressing p150 nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of expression of such DNAs. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector, that upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those with ordinary skill in the art and include those that are replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. For example, DNAs encoding p150 may be inserted into a vector suitable for expression of cDNAs in mammalian cells, e.g. a CMV enhancer-based vector such as pEVRF (Matthias, et al., (1989) NAR 17, 6418).

Particularly useful for practising the present invention are expression vectors that provide for the transient expression of DNA encoding p150 in mammalian cells. Transient expression usually involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector, and, in turn, synthesises high levels of p150. For the purposes of the present invention, transient expression systems are useful e.g. for identifying p150 mutants, to identify potential phosphorylation sites, or to characterise functional domains of the protein.

Construction of vectors according to the invention employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing p150 expression and function are known to those skilled in the art. Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.

In accordance with another embodiment of the present invention, there are provided cells containing the above-described nucleic acids. Such host cells such as prokaryote, yeast and higher eukaryote cells may be used for replicating DNA and producing p150. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, such as E. coli, e.g. E. coli K-12 strains, DH5a and HB101, or Bacilli. Further hosts suitable for p150 encoding vectors include eukaryotic microbes such as filamentous fungi or yeast, e.g. Saccharomyces cerevisiae. Higher eukaryotic cells include insect and vertebrate cells, particularly mammalian cells. In recent years propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are epithelial or fibroblastic cell lines such as Chinese hamster ovary (CHO) cells, NIH 3T3 cells, HeLa cells or 293T cells. The host cells referred to in this disclosure comprise cells in in vitro culture as well as cells that are within a host animal.

DNA may be stably incorporated into cells or may be transiently expressed using methods known in the art. Stably transfected mammalian cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient transfectants, mammalian cells are transfected with a reporter gene to monitor transfection efficiency.

To produce such stably or transiently transfected cells, the cells should be transfected with a sufficient amount of p150-encoding nucleic acid to form p150. The precise amounts of DNA encoding p150 may be empirically determined and optimised for a particular cell and assay.

Host cells are transfected or, preferably, transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique or by electroporation. Numerous methods of transfection are known to the skilled worker in the field. Successful transfection is generally recognised when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used.

Incorporation of cloned DNA into a suitable expression vector, transfection of eukaryotic cells with a plasmid vector or a combination of plasmid vectors, each encoding one or more distinct genes or with linear DNA, and selection of transfected cells are well known in the art (see, e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).

Transfected or transformed cells are cultured using media and culturing methods known in the art, preferably under conditions, whereby p150 encoded by the DNA is expressed. The composition of suitable media is known to those in the art, so that they can be readily prepared. Suitable culturing media are also commercially available.

While the DNAs provided herein may be expressed in any suitable host cell, e.g. those referred to above, preferred for expression of DNA encoding functional p150 are eukaryotic expression systems such as baculovirus-based systems and, particularly, mammalian expression systems, including commercially available systems and other systems known to those of skill in the art.

In preferred embodiments, p150 encoding DNA is ligated into a vector, and introduced into suitable host cells to produce transformed cell lines that express p150. The resulting cell lines can then be produced in quantity for reproducible qualitative and/or quantitative analysis of the effect(s) of potential drugs affecting p150 function. Thus p150 expressing cells may be employed for the identification of compounds, particularly small antagonising molecules interfering with p150 function are useful. An alternative to achieve an antagonistic effect is to rely on over-expression of antisense p150 RNA. Thus host cells expressing p150 are useful for drug screening and it is a further object of the present invention to provide a method for identifying compounds which modulate and preferable reduce the activity of p150, said method comprising exposing cells containing heterologous DNA encoding p150, wherein said cells produce functional p150, to at least one compound or signal whose ability to modulate the activity of said p150 is sought to be determined, and thereafter monitoring said cells for changes caused by said modulation. Such an assay enables the identification of agonists, antagonists and allosteric modulators of p150.

Cell-based screening assays can be designed e.g. by constructing cell lines in which the expression of a reporter protein, i.e. an easily assayable protein, such as b galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase, is dependent on p150. Such an assay enables the detection of compounds that directly modulate p150 function, e.g. compounds that antagonise p150, or compounds that inhibit other cellular functions required for the activity of p150. An in vitro assay for p150 requires that it may be produced in large amounts in a functional form using recombinant DNA methods. An assay is then designed to measure a functional property of the p150.

It has been found that p150 is expressed mostly in cells of high metastatic potential. Thus the present invention also provides a method to exogenously affect p150 dependent processes occurring in such cells. Recombinant p150 producing host cells, e.g. mammalian cells, can be contacted with a test compound, and the modulating effect(s) thereof can then be evaluated by comparing the p150-mediated response in the presence and absence of test compound, or relating the p150-mediated response of test cells, or control cells (i.e., cells that do not express p150), to the presence of the compound.

As used herein, a compound or signal that modulates the activity of p150 refers to a compound that alters the activity of p150 in such a way that the activity of p150 is different in the presence of the compound or signal (as compared to the absence of said compound or signal).

The invention also provides a transgenic non-human mammal which has been modified to modulate the expression of endogenous p150. Preferably, the transgenic non-human mammal is a transgenic mouse. For example, therefore, a transgenic mouse may be designed in which p150 production is greatly reduced or eliminated Alternatively, the transgenic mouse of the invention may express elevated levels of p150, or may be subject to regulation of p150 expression in a developmentally or tissue-specific manner, or via control by exogenous agents. Study of such an animal provides insights into the importance of p150 in vivo.

Moreover, since the polypeptides according to the invention are closely associated with the metastatic process in tumour cells, the invention provides a composition comprising a nucleic acid encoding a polypeptide according to the invention or an antagonist thereto for use as a medicament in the treatment or diagnosis of tumours.

In a preferred embodiment, there is provided a transcription unit encoding a polypeptide according to the invention or an antagonist thereto for use in a method of treatment of a tumour or other condition, for example a condition involving aberrant p150 gene expression, by gene therapy techniques. The transcription unit provided according to the present aspect of the invention comprises regulatable control regions which include a promoter, together with one or more enhancers and/or LCRs. The transcription unit may be delivered to the subject by any suitable means, including viral vectors, especially retroviral vectors, adeno- and adeno associated viral vectors, non-viral delivery systems, including liposomal and antibody targeted delivery systems, and direct uptake of naked DNA. The target tissue is advantageously a tumour tissue.

In accordance with yet another embodiment of the present invention, there are provided antibodies specifically recognising and binding to p150. For example, such antibodies may be generated against the p150 having the amino acid sequences set forth in SEQ ID Nos. 2 and 9, respectively. Alternatively, p150 or p150 fragments (which may also be synthesised by in vitro methods) are fused (by recombinant expression or an in vitro peptidyl bond) to an immunogenic polypeptide and this fusion polypeptide, in turn, is used to raise antibodies against a p150 epitope.

Anti-p150 antibodies are recovered from the serum of immunised animals. Alternatively, monoclonal antibodies are prepared from cells in vitro or from in vivo immunised animals in conventional manner. Preferred antibodies identified by routine screening specifically identify p150 on tumour cells with a high metastatic potential.

The antibodies of the invention are useful for studying p150 tissue localisation, screening of an expression library to identify nucleic acids encoding p150 or the structure of functional domains, as well as for the purification of p150, and the like. Preferably, however, they are useful in the diagnosis and treatment of tumours and especially tumour metastasis.

Antibodies according to the invention may be whole antibodies of natural classes, such as IgE and IgM antibodies, but are preferably IgG antibodies. Moreover, the invention includes antibody fragments, such as Fab, F(ab′)₂, Fv and ScFv. Small fragments, such Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.

The antibodies according to the invention are especially indicated for the diagnosis and therapy of cancer. Accordingly, they may be altered antibodies comprising an effector protein such as a toxin or a label. Especially preferred are labels which allow the imaging of the distribution of the antibody in a tumour in viva. Such labels may be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within the body of a patient. Moreover, the may be fluorescent labels or other labels which are visualisable on tissue samples removed from patients.

Recombinant DNA technology may be used to improve the antibodies of the invention. Thus, chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications. Moreover, immunogenicity may be minimised by humanising the antibodies by CDR grafting [see European Patent Application 0 239 400 (Winter)] and, optionally, framework modification [see international patent application WO 90/07861 (Protein Design Labs)].

Antibodies according to the invention may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture. Preferably, monoclonal antibodies according to the invention are antibody 6G7 (DSM 2256) and antibody 6G10 (DSM 2257). Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or preferable in mammalian cell culture. The selected cell culture system preferably secretes the antibody product.

Therefore, the present invention includes a process for the production of an antibody according to the invention comprising culturing a host, e.g. E. coli or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said protein, and isolating said protein.

Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, e.g. fetal calf serum, or trace elements and growth sustaining supplements, e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like. Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2×YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies. Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilised or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges.

Large quantities of the desired antibodies can also be obtained by multiplying mammalian cells in vivo. For this purpose, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody-producing tumours. Optionally, the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection. After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.

The cell culture supernatants are screened for the desired antibodies, preferentially by immunofluorescent staining of cells expressing p150, by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culture supernatants or in the ascitic fluid may be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like. If necessary and/or desired, the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno)-affinity chromatography, e.g. affinity chromatography with p150 protein or with Protein-A.

The invention further concerns hybridoma cells secreting the monoclonal antibodies of the invention. The preferred hybridoma cells of the invention are genetically stable, secrete monoclonal antibodies of the invention of the desired specificity and can be activated from deep-frozen cultures by thawing and recloning.

The invention also concerns a process for the preparation of a hybridoma cell line secreting monoclonal antibodies directed to p150, characterised in that a suitable mammal, for example a Balb/c mouse, is immunised with purified p150 protein, an antigenic carrier containing purified p150 or with cells bearing p150, antibody-producing cells of the immunised mammal are fused with cells of a suitable myeloma cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example spleen cells of Balb/c mice immunised with cells bearing p150 are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag14, the obtained hybrid cells are screened for secretion of the desired antibodies, and positive hybridoma cells are cloned.

Preferred is a process for the preparation of a hybridoma cell line, characterised in that Balb/c mice are immunised by injecting subcutaneously and/or intraperitoneally between 10 and 10⁷ and 10⁸ cells of human tumour origin which express p150 containing a suitable adjuvant several times, e.g. four to six times, over several months, e.g. between two and four months, and spleen cells from the immunised mice are taken two to four days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol. Preferably the myeloma cells are fused with a three- to twentyfold excess of spleen cells from the immunised mice in a solution containing about 30% to about 50% polyethylene glycol of a molecular weight around 4000. After the fusion the cells are expanded in suitable culture media as described hereinbefore, supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells.

The invention also concerns recombinant DNAs comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to the extracellular domain of p150 as described hereinbefore. By definition such DNAs comprise coding single stranded DNAs, double stranded DNAs consisting of said coding DNAs and of complementary DNAs thereto, or these complementary (single stranded) DNAs themselves.

Furthermore, DNA encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies directed p150 can be enzymatically or chemically synthesised DNA having the authentic DNA sequence coding for a heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof. A mutant of the authentic DNA is a DNA encoding a heavy chain variable domain and/or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids. Preferably said modification(s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody. Such a mutant DNA is also intended to be a silent mutant wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s). Such a mutant sequence is also a degenerated sequence. Degenerated sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded. Such degenerated sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain murine variable domain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and the expression of chimeric antibodies, the recombinant DNA inserts coding for heavy and light chain variable domains are fused with the corresponding DNAs coding for heavy and light chain constant domains, then transferred into appropriate host cells, for example after incorporation into hybrid vectors.

The invention therefore also concerns recombinant DNAs comprising an insert coding for a heavy chain murine variable domain of an antibody directed p150 fused to a human constant domain g, for example g1, g2, g3 or g4, preferably g1 or g4. Likewise the invention concerns recombinant DNAs comprising an insert coding for a light chain murine variable domain of an antibody directed to p150 fused to a human constant domain k or l, preferably k.

In another embodiment the invention pertains to recombinant DNAs coding for a recombinant antibody wherein the heavy chain variable domain and the light chain variable domain are linked by way of a DNA insert coding for a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and/or a DNA coding for a peptide facilitating the purification of the antibody and/or a DNA coding for a cleavage site and/or a DNA coding for a peptide spacer and/or a DNA coding for an effector molecule.

The DNA coding for an effector molecule is intended to be a DNA coding for the effector molecules useful in diagnostic or therapeutic applications. Thus, effector molecules which are toxins or enzymes, especially enzymes capable of catalysing the activation of prodrugs, are particularly indicated. The DNA encoding such an effector molecule has the sequence of a naturally occurring enzyme or toxin encoding DNA, or a mutant thereof, and can be prepared by methods well known in the art.

Antibodies and antibody fragments according to the invention are useful in the diagnosis and therapy of tumours. Accordingly, the invention provides a composition for the therapy or diagnosis of tumours comprising an antibody according to the invention.

In the case of a diagnostic composition, the antibody is preferably provided together with means for detecting the antibody, which may be enzymatic, fluorescent, radioisotopic or other means. These means can e.g. be used to screen specific tissues and body fluids like e.g. blood, serum, ascites, spinal-fluid and urine. The antibody and the detection means may be provided for simultaneous, simultaneous separate or sequential use, in a diagnostic kit intended for the diagnosis of tumour conditions.

The invention further provides a method for determining the metastatic potential of a tumour cell, comprising assessing the level of p150 expression in said cell, a higher level of p150 expression being indicative of a lower state of differentiation and consequently a higher metastatic potential.

Moreover, the invention provides a method for the therapy of tumours comprising administering to a patient a therapeutically effective dose of an antibody, a polypeptide or a nucleic acid according to the invention.

The invention is further described, for the purposes of illustration only, in the following examples.

EXAMPLE 1

Metastasis-specific antibody

Due to the interest in the molecular components playing a role in the metastatic cascade monoclonal antibodies (Mabs) are raised against fractions containing membrane proteins derived from human placenta in order to identify such molecules possibly involved in this complex event.

Membrane-associated proteins are isolated from human placental tissue. The rationale for the selection of placental tissue is that since the trophoblast invades surrounding tissue, it is likely that invasion-associated proteins will be expressed therein.

200 g fresh human placenta is used as a starting material. The placenta is cut into small pieces and then washed several times in ice cold PBS in order to remove blood. The small pieces are transferred into a beaker containing 200 ml lysis buffer (100 mM Octyl-beta-D-glucoside, 1 mM PMSF, 5 mg/ml Leupeptin and 1 mM Mn²⁺ dissolved in PBS), homogenised with a Warren blender, stirred for 4 h at 4° C. and then once again homogenised with a Polytron. Solid matter is then removed by centrifugation at 25000 g for 60 minutes.

The supernatant is applied to a preequilibrated 10 ml column charged with Sepharose® conjugated with the hexapeptide GRGDSP. The column is then extensively washed with lysis buffer. Bound material is eluted in lysis buffer containing 10 mM EDTA. 1 ml eluate fractions are analysed by SDS-PAGE under reducing conditions.

Balb/c mice are then used for raising Mabs against putative RGD-binding proteins. Each animal receives about 50-100 mg of partially purified membrane proteins derived from the Sepharose®-GRGDSP column, plus 0.5 ml Freunds Adjuvants injected ip. As booster applications, the animals twice receive 50-100 mg of protein plus 0.5 ml Freunds incomplete Adjuvants every 3-4 weeks. Finally, 2 days before the fusion of the splenocytes with the myeloma cells ( PAI cells), the animals receive another 20-30 mg of protein intravenously. The resulting Mabs are screened for cell surface staining and expression levels of the newly defined antigens in transformed versus non transformed cells. Comparison is made between 3T3 cells in a non-transformed state, and 3T3 cells transformed with src or ras, as well as CEF68 cells grown at permissive or non-permissive temperatures.

Cell surface staining is analysed by immunofluorescence, as follows. Cells are grown on glass coverslips or on glass slides, washed in PBS or in DME and fixed for 30 min. in formaldehyde 3.7% in PBS or in DME at room temperature (20° C.). Cells are then permeabilised in 0.5% TX-100 (in PBS or DME) for 1-3 min at room temperature. Subsequently, cells are washed 3-4 times in PBS or DME at room temperature. Liquid is drained off and 0.03 ml of a first antibody raised against the membrane-associated protein fractions diluted in DME/10% FCS is added. The cells are incubated for 60 minutes at 37° C. in a humid incubator.

After incubation, the cells are washed 3-4 times in PBS or DME at room temperature. 0.03 ml of a second, anti-mouse antibody labelled with FITC or TRITC diluted in DME /10% FCS is added, and the cells incubated for 60 minutes at 37° C. in a humid incubator. Subsequently, they are washed 3-4 times in PBS or DME and once in water. They are then embedded in an appropriate mounting medium and stored in the dark. Fluorescence can then be assessed by viewing the embedded cells using suitable fluorescence microscopy equipment.

An interesting MAb, H11VII, is selected and further tested in the B16 F1/F10 mouse melanoma system. For this purpose melanoma cells are incubated with the MAb prior to tail vein injection into mice. After 3 weeks the animals are sacrificed and the lung colonies counted. A 80-85% inhibition of the lung colonisation ability can be observed.

In order to reconfirm the results obtained with the original MAb (H11VII) a new set of Mabs against mouse p150 is raised. The outcome of the in vivo lung colonisation assay performed with the new anti-p150 Mabs is shown in Table 1.

TABLE 1 B16 F1 cells treatment number of lung colonies mean std % inhibition control 25,16,2,1,14,20,25,24 16 10 H11VII 1,0,5,9,0,4,0,0,5,9 3 4 80 B16 F10 cells treatment number of lung colonies mean std % inhibition untreated 36,45,23,20,52,64, 46 8 >300(64), >300(64) control 27,22,38,22,20,28,37,64, 44 29 93,>300(93) H11VII 0,0,0,1,3,4,10,11,12,16 6 6 85 B16 F1 cells H11VII induced treatment number of lung colonies mean std fold induc. control 2,6,5,5,13,0,3,1,2,35 4 4 H11VII ind 0h 28,97,92,37,61,7,43,17, 43 32 10 5,47 H11VII ind 40h 41,45,47,74,21,26,51,127, 66 40 16 101,131 B16 F1 cells treatment number of lung colonies mean std % inhibition 4H9 0,3,>50,>50,22,>50,20 ? ? promotion? 6G7 1,9,4,9,2,1,4,3,8,0 4 3 80 H11VII 11,1,13,11,0,7,16 8 5 50 control 22,33,24,7,19,30,4,34 22 11 1B10 25,1,13,13,0,7,8,16,10 10 8 50 6G10 9,6,3,4,7,2,2,7,23,0 6 6 70

A general problem by using antibodies in an in vivo system is the possible stimulation of the host immune system. Especially the cytotoxic as well as the natural killer activities towards cells tagged with antibodies can be influenced. Due to the fact that peripheral cellular blood components do not express p150 in detectable amounts, it is possible to reduce the influence of the host immune system by changing the treatment. Instead of incubation of the cancer cells with Mabs prior to injection we pretreat the host animals 24 hrs before they received F1 cells. The results of this experiment, in which the original MAb (H11VII) as well as a new MAb raised against mouse p150 (6G7) are used, are outlined in Table 2.

TABLE 2 B16 F1 cells injected into pretreated animals pretreatment number of lung colonies mean std % inhibition control 1,4,13,31,40,44,63,>99(63), 39 25 n/a >99(63),>99(63) H11VII 1,1,5,7,10,15,17,25,41 14 13 65 6G7 0,1,2,5,6,11,19,20,22 10 9 75

Mice are pretreated with the monoclonal antibodies for 24 hours before injection of B16 cells. 0.5 mg of antibody is injected i.p. Antibodies are prepared by precipitation in 28 g/100 ml (NH₄)₂SO₄ and ConA chromatography.

EXAMPLE 2

Metastasis-specific antigen

Using western blotting according to standard protocols, antibody H11VII is seen to recognise bands on SDS-PAGE migrating at approximately 28, 35, 70 and 150 KDa. The bands are purified from the gel and rabbit polyclonal antisera raised thereto. The antiserum raised against the 28 KDa band cross-reacts with the 150 KDa band on western blots.

The antigen recognised by MAb H11VII can be identified as a 150 kDa membrane associated protein (p150). P150 is expressed in B16F1 cells in large amounts. Therefore, these cells are used as starting material for the purification of p150.

A crude membrane fraction is isolated from B16F1 cells by centrifugation at 100,000 g and lysis in 1%NP40, 2 mM Tris-HCl pH 7.5, containing PMSF and aprotinin protease inhibitors. The supernatant is loaded on a Sepharose® column, run off, transferred to a Sepharose® S column, again run off, and separated by reducing SDS-PAGE on a 5% gel. The 150 KDa band is electrocuted.

EXAMPLE 3

Analysis of p150 and cDNA cloning

P150 is microsequenced by digestion with trypsin, V8 and Lys-C followed by sequencing using Edman degradation on an Applied Biosystems sequencer. An oligonucleotide deduced from the sequence used to probe a mouse melanoma B16F1 library.

Sequencing the clones picked out of the B16F1 mouse melanoma c-DNA library identifies an ORF of 3.6 kb encoding the 142 kDa carboxy terminal part of p150. The missing 5′ part was obtained by applying race technique using a kit from Clontech. Taking all the sequence information together, we can establish an ORF of 4.03 kb encoding a polypeptide of 1343 aa. This ORF, referred to as p150 is shown in SEQ ID No. 1. The deduced protein sequence is shown in SEQ ID No. 2 and FIG. 1. The peptide sequences obtained from protein microsequencing are underlined in FIG. 1. The cDNA and derived amino acid sequences of the human homologue, also referred to as p150 are shown in SEQ ID Nos. 8 and 9, respectively.

Striking homologies are found between p150 and two mouse proteins called centrosomin A (280 amino acids from the sequence shown in SEQ ID No. 3) and B (430 amino acids from the sequence shown in SEQ ID No. 4) respectively. These two proteins are most likely splice variants. They are found to be associated with centrosomes. Unfortunately no functional studies have been carried out on centrosomins, but, due to the cellular localisation, they are thought to take part in the organisation of the spindle apparatus and therefore could be candidates for possible tubulin binding.

Sequence alignment between p150, centrosomin A and centrosomin B shows that the centrosomins share close homology with p150 throughout their lengths, but p150 extends both 5′ and 3′ of both centrosomins. A sequence comparison is given in FIG. 2.

Another 3 ORFs showing significant homologies to p150 exist in the Swiss Prot databank. A yeast ORF (YBR079; SEQ ID No. 5) showing 51% similarity and 29% identity, a tobacco ORF (NTPNL35; SEQ ID No. 6) with 59% similarity and 37% identity and an ORF found in nematode C.elegans (EGL-45; SEQ ID No. 7) showing 56% similarity and 36% identity. Unfortunately functional data are available only for EGL-45; the two other ORFs are of unknown function. Mutational studies performed on EGL-45 suggest an involvement of this protein in the regulatory steps of the hermaphrodite specific neuronal differentiation. Expression of EGL-45 is found in most cells of the worm. Null mutants give rise to a sterile or even a lethal phenotype.

All four sequences show a common overall organisation: namely a stretch of 300 aa at the amino terminus with significant identities between 30-45%, followed by a stretch of about 200aa of little homology which is followed by a highly charged stretch of about 400aa predicted to form coiled-coil domains (according to Lupas et al., Science 252, 1162, 1991).

The sequence alignments of 6 proteins described hereinabove (including the centrosomins) are shown in FIG. 2.

EXAMPLE 4

Metastasis-specific expression of p150

It is reasoned that p150 on B16F1/F10 cells must be expressed on the cell surface, otherwise it would not be possible to influence the metastatic cascade by applying Mabs to the cell surface. In order to verify the localisation of p150, FACS analysis is carried out as follows:

Starting with a confluent 6 cm tissue culture dish with B16 F1 or F10 cells (in DME-medium), antibody (dilution 1/10-1/100) is added to adherent cells or detached cells (EDTA, PBS w/o Ca²⁺/Mg²⁺, wash once in PBS/1% BSA, spin down and add the antibody).

Cells are incubated either on the tissue culture plate (at 20 or 37° C.) or in an Eppendorf tube for 1-2 h. Subsequently, cells are washed with PBS/1% BSA on the plate and detached, or washed by spinning down in the Eppendorf tube and adding PBS/1% BSA. The washing step is repeated 3-4 times.

Cells which were labelled on the plate are then taken up in 1 ml PBS/1% BSA.

A second antibody conjugated with FITC or TRITC is added to the cells in PBS/1% BSA and incubated for 1 h at 20° C. while slowly shaking. The cells are washed 2-3 times with PBS/1% BSA. A FACscan may then be carried out according to standard protocols.

FACS analysis demonstrates that P150 is clearly expressed at the cell surface of B16 F1 mouse melanoma cells. The labelling pattern as well as the intensity of the staining is independent of the fact whether the cells were labelled in suspension or they were attached to tissue culture dishes.

Results are confirmed by cell surface biotinylation on living F1 cells, as follows:

A confluent 15 cm dish of B16 F1 or HeLa cells is washed 3-4 times with cold PBS. 10 ml PBS containing 0.05 ml Biotinamidocaproate-N-Hydroxysuccimide Ester (Sigma B 2643), which was dissolved in DMSO at 100mg/ml, is then added and the cells incubated for 40-60 minutes in the cold room (4° C.).

The cells are then washed 2-3 times with cold PBS and lysed in cold lysis buffer (see p150 purification) for 30 minutes on ice. Undissolved material is spun down at 13000 g for 10 minutes and the cleared cell lysate incubated with Avidin Agarose beads for 30 minutes on a shaker in the cold room.

The Avidin Agarose beads are washed with lysis buffer 3-4 times, once with cold ddH₂O and boiled in SDS-PAGE sample buffer. SDS-PAGE is run and western blotting carried out according to standard protocols.

B16 F1 cells are seen to express p150 on the cell surface as determined by the above cell surface biotinylation method. However only a very small proportion of total cellular p150 is exposed at the cell surface. This seems to be involved in cell-cell and/or in cell-extracellular matrix interactions as already indicated by the results obtained from the in vivo colonisation experiments. Contrary to this finding in B16 cells, in HeLa cells no cell surface biotinylation of p150 could be observed.

According to its amino acid sequence, p150 contains a hydrophobic stretch of 70 aa (1050-1120), which could serve as a transmembrane domain. However, most of the cellular p150 is found in the cytoplasmic compartment associated with endogenous membranes.

On HeLa cells, in contrary to the B16F1 cells, p150 could not be detected on the cell surface by biotinylation technique. Therefore, it might be possible, that the distribution of p150 depends on the cell type and on its status.

B16 cells show a strong transformed phenotype; e.g. they do not spread out very well in cell culture. As a consequence, the cytoskeletal elements are not as well separated as in cells which show a more spread out phenotype. Therefore we chose HeLa cells for our localisation studies. Using confocal microscopy, a strong perinuclear staining is observed, while throughout the cytoplasm staining is associated with vescicular structures. In cells undergoing mitosis, staining is observed to be associated with the midbody, in a pattern similar to that of tubulin.

Assessed by western blotting according to standard procedures, p150 is normally expressed in several mouse organs. We find a high expression level in pancreas, as well as liver, although signal in liver might be artefact due to high protein concentration in that region, and a medium level in brain, thymus, and testis. Low expression of p150 occurs in lung, spleen and kidney. P150 expression in testis could reflect the fact that there we can find a lot of undifferentiated, proliferating cells. In testis, p150 and b-tubulin codistribute on structures resembling sperm tails.

In order to analyse the connection between transformation and p150 expression, we analyse various cell types in different states of transformation using western blotting and find that a positive correlation between the state of transformation and the expression level of p150 can be observed. Thus, we observe higher levels of p150 in 3T3 cells transformed with ras than in non-transformed cells, and much higher levels in 3T3 cells transformed with v-src. CEF cells transformed with a temperature sensitive transforming gene product, NY68, show higher levels of p150 expression at the permissive (37° C.) than the non-permissive (41° C.) temperature.

When the expression of p150 assessed by western blotting is correlated to differentiation state, an inverse relationship between the grade of differentiation and the expression level of p150 is observed in all cell lines so far investigated, including ES cells differentiated according to standard procedures, stably differentiated F9 cells and B16F1 cells treated with retinoic acid (0.01 and 0.1 M).

The expression of p150 correlates inversely with malignancy in different B16 variants. The two most malignant variants, F10 and LS9, have a lower level of p150 expression than F1 cells. This seems to contradict the relationship between transformation state and p150 expression levels. However, it is known that in the B16 system the metastatic ability in most cases correlates directly with differentiation state. Thus, in B16 cells, the more differentiated cells are more malignant. This is in contradistinction to what is found in other model systems and in clinics where greater malignancy and poor prognosis correlates inversely with differentiation state.

In order to examine the expression of p150 in other systems, several human tumour cell lines are tested for p150 expression. Some breast cancer cell lines (HBL 100, BT 20, T47D, SKBR3, MB231 and MB453), a liver cancer cell line (Hep G2) as well as a ovarian cancer cell line (SKO V3) are investigated. The p150 levels are different in these lines, but p150 appears to be universally present.

EXAMPLE 5

Expression of p150 in human tumours

Methods

Tumor and normal tissue samples

From 22 fresh mastectomy specimen, cancer tissue and non-cancerous normal tissue is chosen macroscopically by an experienced pathologist for transfer to the laboratory. The tissue arrives in the laboratory about 5 hours after mastectomy at latest, is frozen and stored at −80° C. Corresponding cancer and normal tissue are fixed in 10% buffered formalin and embedded in paraffin. 5 micron sections are cut for hematoxylin/eosin staining. For immunohistochemistry the 5 micron sections are mounted on cementit (Merz und Benteli SA, Niederwangen, Switzerland) covered super frost slides. Presence of invasive cancer and of normal tissue is confirmed by histology in each case.

Preparation of tissue extracts

Frozen human tumor and normal tissue is cut into small pieces, suspended in STE buffer (0.1 M NaCl, 0.01 M Tris-HCl pH 7.6, 1 mM EDTA) with protease inhibitors aprotinin 2 μg/ml, leupeptin 1 μg/ml and 1 mM PMSF and homogenized with a Polytron homogenizer. The homogenates are centrifuged at 1000 g for 15 minutes and the supernatants are mixed with one volume of 2×sample buffer consisting of 0.1 M Tris-HCl pH 6.8, 4% SDS and 20% glycerol. This mixture is incubated for 30 minutes at 56° C. and recentrifuged at 15,000 g for 10 minutes. The protein concentration of the supernatants is determined with a BCA protein assay kit (Pierce, Rockford, Ill.).

Preparation of NP-40 extracts of cell lines

Cells are washed twice with PBS and lysed in ice-cold lysis buffer (0.01 mM Tris-HCl pH 7.5, 0.5% Nonidet P-40 and 1 mM PMSF) in the dish for 30 minutes at 4° C. The lysed cells are centrifuged at 15,000 g at 4° C. The supernatants are mixed with sample buffer as described above. The samples are heated for 2 minutes at 95° C. and the protein concentrations determined.

Immunoblotting

Extracts of cells or surgical specimens containing 20 or 40 μg of protein are subjected to electrophoresis in the presence of 0.1% SDS in 5% SDS-PAGE minigels. After electrophoresis, the proteins are transferred electrophoretically at 1 mA per cm² for 90 minutes onto a PVDF membrane. The membranes are blocked in blocking solution (0.1 M Tris-HCl pH 7.4, 0.1 M MgCl₂, 0.5% Tween 20, 1% Triton X-100, 1% BSA, 5% FCS) for 30 minutes at room temperature (RT), decorated with a chicken Ab against p150 (Sophie 1:2000) or with Mab 6G10 (supernatant 1:20) overnight at 4° C., washed with blocking solution and then incubated for 1 hour at RT with a goat anti-chicken peroxidase-coupled second antibody (1:2000, Southern Biotechnology Ass. Inc., Birmingham, Ala.) or rabbit anti-mouse and swine anti-rabbit (both peroxidase-coupled, Dako Diagnostics AG, Zug, Switzerland) at dilutions of 1:2000. Then the membranes are washed with blocking solution, PBS and water and the protein-antibody complexes are visualized by ECL (Amersham International, UK) according to the manufacturer's protocol. Quantification of the Western blots are done by using a ¹²⁵Iodine-labeled second antibody (Amersham International, UK). The blots are scanned with a Phosphorimager unit (Molecular Dynamics, Sunnyvale, Calif.) and the relative amounts of p150 are determined by measuring the radioactivity present in the appropriate bands.

Immunohistochemistry

Representative tissue blocks of normal and carcinoma tissue are taken from three cases for immunohistological examination. The 5 micron paraffin sections are deparaffinized, rehydrated and washed twice in Tris-NaCl buffer (30 mM Tris (pH 7.5), 0.9% NaCl). Internal peroxidases are blocked with blocking solution (25 mM β-D-glucose, 25 mM sodium azide and 0.6 units/ml glucose oxidase (Sigma, Switzerland) in Tris-NaCl buffer) for 1 hour at 37° C. After washes in Tris-NaCl buffer, the sections are heated in 500 ml citrate buffer (0.1 mM, pH 6.0) in the microwave oven operated at 750 W for 10 minutes. The sections are allowed to cool down and washed again in Tris-NaCl buffer before blocking with 1% BSA in Tris-NaCl buffer for 15 minutes. The chicken antibody against p150 (Sophie 1/1000) is applied for 60 minutes at RT in the same buffer, whereas the Mab 6G7, the monospecific rabbit anti- NH₂ terminal peptide (225 amino acids) antibody and the Mab against Ki-67 (MIB1, Dianova GmbH, Germany) are incubated with the sections overnight at 6° C. Subsequently the sections are washed twice, incubated with second antibodies (goat anti-chicken, goat anti-mouse or goat anti-rabbit; all peroxidase coupled, diluted 1:2000, Southern Biotechnology Ass. Inc., Birmingham, Ala.) for half an hour, rinsed with Tris-NaCl-buffer and incubated with the peroxidase substrate 3′-3′-diaminobenzidine (Sigma, Switzerland) 2% in 50 mM Tris pH 7.6 for 5 to 10 minutes at RT. The paraffin sections are counterstained with stabilized hematoxylin (Shandon, Astmoore, GB), dehydrated, mounted in Depex (BDH, Gurr, UK) and photographed.

A. Screening of 22 mammary carcinoma

Expression of p150 in human mammary tumors

Based on the fact that transformation and dedifferentiation are main features of cancer cells primary human tumors are screened for p150 expression using the panel of antibodies developed in the early stage of these studies.

The mean age of our 22 patients with primary breast cancer when operated was 64 years (range 37-90 y) and the mean tumor size was 48 mm (range 20-140 mm). 55% of the tumors were poorly differentiated (GIII according to the Elston and Ellis grading system (12)) while 14% were well differentiated. The predominant histological type (91%) was ductal invasive, the others were lobular invasive tumors. Metastasis to axillary lymph nodes was detected in 59%, in 27% the lymph nodes showed no metastasis and from all other patients no lymph nodes were available. In 21 of the 22 patients Western blotting have revealed significantly higher p150 expression in tumors than in the normal tissue counterparts of the same patients. With patient #14139 we have been able to investigate lymphnode metastasis; p150 expression in this context is even higher than in the primary carcinoma. One case (#10594) displayed an aberrant result. This patient, however, was the only one who had received preoperative chemotherapy and did not show any p150 overexpression in the cancer tissue.

Immunohistochemistry

A distinct pattern of p150 expression is observed in breast cancer tissue versus the control tissue. Invasive and in situ ductal cancer react equally positive. Necrotic tissue is found to be negative for p150 expression.

Normal non proliferating tissue shows faint focal cytoplasmic staining of luminal epithelial cells as do myoepithelial cells. A few nuclei in proliferating epithelial cells also present positive staining. Stromal tissue is negative for p150 in all cases.

Proliferative and precancerous alterations in breast tissue such as epitheliosis, apocrine metaplasia and adenosis show staining for p150. No correlation between Ki-67 staining and p150 expression in normal breast tissue is observed.

B. Screening of additional 31 mammary carcinoma

The mean age of the 31 patients with primary breast cancer when operated was 61 years (range 36-85 years) and the mean tumor size was 28 mm (range 9-90 mm). Fifty-five percent of the cancers were poorly differentiated (GIII according to the Elston and Ellis grading system), 32% were moderately differentiated and 13% were benign. The predominant histological type (67%) was ductal invasive, the others were lobular invasive (16%), fibroadenomas (10%), one squamous cell carcinoma, one a mucinous carcinoma and one was a papilloma.

Metastasis to lymphnode was detected in 42%, in 29% no lymph nodes were involved and in 29% of the patients lymph nodes were not available. Thirty of 31 patients show significantly higher p150 expression in tumors than in normal tissue counterparts in Western blot analysis.

One patient (#18976) shows no p150 expression neither in the normal breast tissue nor in the carcinoma. By histology this tumor is determined to be a rare squamous cell carcinoma of the breast. Interestingly, squamous cell carcinomas of the cervix show almost a loss of p150 expression as soon as the tumor starts to invade. Therefore, it is suggested, that the behavior in p150 expression is strongly dependent on the histological type and origin of the particular tumor.

Remarkably, p150 expression in the benign fibroadenomas and papilloma is much lower than in malignant mammary carcinoma.

C. Screening of cervical cancer

Extensive studies have been carried out on squamous cell carcinoma of the cervix. Specimens of 165 patients have been investigated by immunohistochemistry. According to histological grading they have been divided into 3 groups containing 77 cases with precancerous lesions, 68 cases with invasive tumors and 20 healthy controls. The precancerous lesions significantly present the highest level of p150 expression whereas invasive stages show expression levels comparable to controls. Furthermore 120 month survival is significantly increased (p<0.01) in those patients who show highest expression levels of p150.

In contrast to mammary carcinoma, malignancy in cervical squamous cell carcinoma inversely correlates with p150 expression levels. Apparently, the behavior of neoplastic tissue in terms of p150 expression strongly depends on the histological origin. Therefore, it is not a general issue for p150, that expression levels increase with enhanced proliferative ability. Thus it appears that p150 can be used both as a dedifferentiation and as a proliferation marker.

D. Screening of colon cancer

Nine cases of colon cancer are investigated by Western blot analysis. p150 is clearly upregulated in all nine cases in comparison with the control tissue. An additional band of about 120 kD which is considered to be a degradation product, appears exclusively in the carcinoma. This additional band is not recognized by an antibody against the NH₂-terminal peptide of p150. This implies that in colon carcinoma the amino-terminal part of p150 might be specifically degraded by high protease activity.

Immunohistochemistry, which is done on 120 cases including the 9 cases presented in Western blot analysis, clearly reconfirms our findings. Furthermore, the results suggest that p150 plays a role in the dedifferentiation process in colorectal carcinoma.

E. Screening of lung and thyroid cancer

Several cases of lung and thyroide cancer are investigated as well. The data suggest an upregulation of p150 in these malignancies too.

F. Screening of oesophagal cancer

Several cases of oesophagal cancer are investigated as well. From 70 cases 30 are screened by Western blot analysis and 40 by immunohistochemistry. All tissue samples screened with either method show positive results.

Deposit Data:

Antibodies according to the invention have been deposited in accordance with the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Maschenroder Weg 1b, D-3300 Braunschweig, Germany, under the following accession numbers:

Antibody Accession No. Deposit Date 6G7 DSM 2256 Apr. 11, 1996 6G10 DSM 2257 Apr. 11, 1996

9 1 5137 DNA Mus sp. CDS (128)..(4156) 1 gccggctggg cgcgggcatc tgctggcgag gcgcgtggaa gctcgcgcta gttcccctcc 60 gccttctctc ccggtccagg ccactaggga gttcgctgac gccgggtgaa ctgagcgtac 120 cgtcaag atg ccg gcc tat ttc cag agg ccg gaa aat gcc ctc aag cgc 169 Met Pro Ala Tyr Phe Gln Arg Pro Glu Asn Ala Leu Lys Arg 1 5 10 gcc aac gaa ttt ctt gag gtt ggc aaa aag cag cct gcc ctg gat gtt 217 Ala Asn Glu Phe Leu Glu Val Gly Lys Lys Gln Pro Ala Leu Asp Val 15 20 25 30 ctt tat gat gta atg aaa agt aag aag cac aga aca tgg cag aag ata 265 Leu Tyr Asp Val Met Lys Ser Lys Lys His Arg Thr Trp Gln Lys Ile 35 40 45 cat gag cca att atg ctg aaa tac ttg gaa ctg tgt gtg gat ctt cgt 313 His Glu Pro Ile Met Leu Lys Tyr Leu Glu Leu Cys Val Asp Leu Arg 50 55 60 aag agc cac ttg gct aag gaa ggg tta tat caa tat aag aac ata tgc 361 Lys Ser His Leu Ala Lys Glu Gly Leu Tyr Gln Tyr Lys Asn Ile Cys 65 70 75 caa cag gta aac att aaa tct tta gaa gat gtt gtt agg gca tat ttg 409 Gln Gln Val Asn Ile Lys Ser Leu Glu Asp Val Val Arg Ala Tyr Leu 80 85 90 aaa tta gca gag gaa aaa aca gaa gct gct aaa gaa gag tcc caa caa 457 Lys Leu Ala Glu Glu Lys Thr Glu Ala Ala Lys Glu Glu Ser Gln Gln 95 100 105 110 atg gtg tta gat ata gaa gat ctg gat aat att cag act cct gag agt 505 Met Val Leu Asp Ile Glu Asp Leu Asp Asn Ile Gln Thr Pro Glu Ser 115 120 125 gtt ctc tta agt gca gta agt ggg gaa gat act cag gat cgt act gac 553 Val Leu Leu Ser Ala Val Ser Gly Glu Asp Thr Gln Asp Arg Thr Asp 130 135 140 aga ttg cta ctg act ccc tgg gtc aag ttc ctg tgg gat cat aca ggc 601 Arg Leu Leu Leu Thr Pro Trp Val Lys Phe Leu Trp Asp His Thr Gly 145 150 155 agt gtt ttg gac ctt ctt cga aac aat tct aga gta gag cgc ctt tac 649 Ser Val Leu Asp Leu Leu Arg Asn Asn Ser Arg Val Glu Arg Leu Tyr 160 165 170 cat gat atc gcc caa caa gct ttc aaa ttc tgc ctc cag tat act cgg 697 His Asp Ile Ala Gln Gln Ala Phe Lys Phe Cys Leu Gln Tyr Thr Arg 175 180 185 190 aag gct gag ttc cgc aag cta tgt gac aac ttg cga atc gac tta tcc 745 Lys Ala Glu Phe Arg Lys Leu Cys Asp Asn Leu Arg Ile Asp Leu Ser 195 200 205 cag att cag cgc cac cat aac caa agc aca gca att aat ctt aat aat 793 Gln Ile Gln Arg His His Asn Gln Ser Thr Ala Ile Asn Leu Asn Asn 210 215 220 cca gag agc cag tct atg cat ttg gaa acc aga ctt gtt cag ttg gac 841 Pro Glu Ser Gln Ser Met His Leu Glu Thr Arg Leu Val Gln Leu Asp 225 230 235 agt gct atc agc atg gaa tta tgg cag gaa gcc ttc aaa gct gtg gaa 889 Ser Ala Ile Ser Met Glu Leu Trp Gln Glu Ala Phe Lys Ala Val Glu 240 245 250 gat att cat gga cta ttt tcc ttg tct aag aaa cca cct aag cct cag 937 Asp Ile His Gly Leu Phe Ser Leu Ser Lys Lys Pro Pro Lys Pro Gln 255 260 265 270 ttg atg gca aat tac tat aac aaa gtt tca aca gtg ttt tgg aaa tct 985 Leu Met Ala Asn Tyr Tyr Asn Lys Val Ser Thr Val Phe Trp Lys Ser 275 280 285 gga aat gct ttg ttc cat gca tct aca ctt cat cgt ctt tat cat ctg 1033 Gly Asn Ala Leu Phe His Ala Ser Thr Leu His Arg Leu Tyr His Leu 290 295 300 tct aga gaa atg aga aag aat ctt aca caa gaa gag atg caa aga atg 1081 Ser Arg Glu Met Arg Lys Asn Leu Thr Gln Glu Glu Met Gln Arg Met 305 310 315 tct act aga gtc ctt ttg gcg act ctt tcc att cct att act cct gag 1129 Ser Thr Arg Val Leu Leu Ala Thr Leu Ser Ile Pro Ile Thr Pro Glu 320 325 330 cga act gat att gct cga ctt ttg gat atg gat ggt att ata gtt gag 1177 Arg Thr Asp Ile Ala Arg Leu Leu Asp Met Asp Gly Ile Ile Val Glu 335 340 345 350 aag cag cga cgc ctt gcc aca ttg cta ggt ctt caa gcc cca ccc aca 1225 Lys Gln Arg Arg Leu Ala Thr Leu Leu Gly Leu Gln Ala Pro Pro Thr 355 360 365 cga atc ggc cta att aat gat atg gtc agg ttc agt gtg cta cag tat 1273 Arg Ile Gly Leu Ile Asn Asp Met Val Arg Phe Ser Val Leu Gln Tyr 370 375 380 gtt gtc cca gaa gtg aaa gac ctt tac aac tgg ttg gag gtg gaa ttc 1321 Val Val Pro Glu Val Lys Asp Leu Tyr Asn Trp Leu Glu Val Glu Phe 385 390 395 aac cca cta aaa ctc tgt gag aga gtt aca aag gta tta aat tgg gtt 1369 Asn Pro Leu Lys Leu Cys Glu Arg Val Thr Lys Val Leu Asn Trp Val 400 405 410 agg gaa caa cct gaa aaa gaa cca gaa ttg caa caa tat gta cca caa 1417 Arg Glu Gln Pro Glu Lys Glu Pro Glu Leu Gln Gln Tyr Val Pro Gln 415 420 425 430 ctc cag aac aat acc ata ctc cgc ctt ctg caa cag gtg gca cag att 1465 Leu Gln Asn Asn Thr Ile Leu Arg Leu Leu Gln Gln Val Ala Gln Ile 435 440 445 tat cag agc att gag ttt tct cgt ttg act tct ctg gtt cct ttt gtt 1513 Tyr Gln Ser Ile Glu Phe Ser Arg Leu Thr Ser Leu Val Pro Phe Val 450 455 460 gat gct ttc caa ctg gaa cgg gcc ata gta gat gca gcc agg cac tgt 1561 Asp Ala Phe Gln Leu Glu Arg Ala Ile Val Asp Ala Ala Arg His Cys 465 470 475 gac ctg cag gta cgt att gac cat act tcc cgg act ctg agt ttt gga 1609 Asp Leu Gln Val Arg Ile Asp His Thr Ser Arg Thr Leu Ser Phe Gly 480 485 490 tca gat ttg aat tat gca act cga gaa gat gcc cca gtt ggc cct cat 1657 Ser Asp Leu Asn Tyr Ala Thr Arg Glu Asp Ala Pro Val Gly Pro His 495 500 505 510 ctg cag agc atg cct tca gag cag ata aga aac cag ctc acg gcc atg 1705 Leu Gln Ser Met Pro Ser Glu Gln Ile Arg Asn Gln Leu Thr Ala Met 515 520 525 tcc tca gtg ctt gcc aaa gca ctt gaa gtc atc aga cct gct cac att 1753 Ser Ser Val Leu Ala Lys Ala Leu Glu Val Ile Arg Pro Ala His Ile 530 535 540 ctg caa gag aaa gaa gaa cag cat caa ttg gca gtt aat gca tat ctt 1801 Leu Gln Glu Lys Glu Glu Gln His Gln Leu Ala Val Asn Ala Tyr Leu 545 550 555 aaa aat tca aga aaa gag cac cag agg atc ctg gct cgg aga cag aca 1849 Lys Asn Ser Arg Lys Glu His Gln Arg Ile Leu Ala Arg Arg Gln Thr 560 565 570 att gag gaa aga aaa gag cgt ctt gag agt ctg aat att caa cgt gag 1897 Ile Glu Glu Arg Lys Glu Arg Leu Glu Ser Leu Asn Ile Gln Arg Glu 575 580 585 590 aag gaa gaa ctc gag cag agg gaa gct gaa ctc cag aag tac gaa agg 1945 Lys Glu Glu Leu Glu Gln Arg Glu Ala Glu Leu Gln Lys Tyr Glu Arg 595 600 605 ctg aag aag aaa ggc tgc caa gag gca aag gag cga gag aag gaa cga 1993 Leu Lys Lys Lys Gly Cys Gln Glu Ala Lys Glu Arg Glu Lys Glu Arg 610 615 620 atc ctt caa gaa cac gag caa atc aag aag aaa act gtt cgg gag cgg 2041 Ile Leu Gln Glu His Glu Gln Ile Lys Lys Lys Thr Val Arg Glu Arg 625 630 635 tta gag cag atc aag aag aca gag ctg ggc gcc aaa gca ttt aaa gat 2089 Leu Glu Gln Ile Lys Lys Thr Glu Leu Gly Ala Lys Ala Phe Lys Asp 640 645 650 att gac att gaa gac ctt gaa gaa ctg gat cca gat ttt att atg gcc 2137 Ile Asp Ile Glu Asp Leu Glu Glu Leu Asp Pro Asp Phe Ile Met Ala 655 660 665 670 aaa cag gtt gaa caa ctg gag aaa gaa aag aag gaa ctt cag gaa cgc 2185 Lys Gln Val Glu Gln Leu Glu Lys Glu Lys Lys Glu Leu Gln Glu Arg 675 680 685 ctg aag aat caa gaa aag aag att gac tat ttt gag aga gct aag cgt 2233 Leu Lys Asn Gln Glu Lys Lys Ile Asp Tyr Phe Glu Arg Ala Lys Arg 690 695 700 ttg gaa gaa att cct tta ata aag agt gct tat gag gaa caa agg gtt 2281 Leu Glu Glu Ile Pro Leu Ile Lys Ser Ala Tyr Glu Glu Gln Arg Val 705 710 715 aaa gac atg gac ctg tgg gaa cag caa gaa gaa gaa aga atc act aca 2329 Lys Asp Met Asp Leu Trp Glu Gln Gln Glu Glu Glu Arg Ile Thr Thr 720 725 730 atg cag cta gaa cga gaa aaa gct ctg gag cat aag aat agg atg tca 2377 Met Gln Leu Glu Arg Glu Lys Ala Leu Glu His Lys Asn Arg Met Ser 735 740 745 750 cga atg ttg gaa gac aga gat cta ttt gtg atg cgc ctc aaa gct gcc 2425 Arg Met Leu Glu Asp Arg Asp Leu Phe Val Met Arg Leu Lys Ala Ala 755 760 765 cgg cag tct gtc tac gag gaa aaa ctg aaa cag ttt gaa gag cgc tta 2473 Arg Gln Ser Val Tyr Glu Glu Lys Leu Lys Gln Phe Glu Glu Arg Leu 770 775 780 gca gaa gaa agg cat agt cgc cta gaa gat cgg aaa agg cag cgg aaa 2521 Ala Glu Glu Arg His Ser Arg Leu Glu Asp Arg Lys Arg Gln Arg Lys 785 790 795 gaa gaa cgc aaa ata act tat tac aga gaa agg aag aag aag agc aga 2569 Glu Glu Arg Lys Ile Thr Tyr Tyr Arg Glu Arg Lys Lys Lys Ser Arg 800 805 810 gga ggg cag agg agc aga tgc tca aag acg gag aag aaa gag aac gtg 2617 Gly Gly Gln Arg Ser Arg Cys Ser Lys Thr Glu Lys Lys Glu Asn Val 815 820 825 830 ctg aga gag caa aac gcg gag gaa gaa ctt aga gag tat cag gag cga 2665 Leu Arg Glu Gln Asn Ala Glu Glu Glu Leu Arg Glu Tyr Gln Glu Arg 835 840 845 gtc aag aaa cta gaa gaa gta gaa agg aaa aag cgg caa aga gag ctg 2713 Val Lys Lys Leu Glu Glu Val Glu Arg Lys Lys Arg Gln Arg Glu Leu 850 855 860 gaa att gaa gaa agg gaa agg cgc aga gag gaa gaa agg aga ctt ggt 2761 Glu Ile Glu Glu Arg Glu Arg Arg Arg Glu Glu Glu Arg Arg Leu Gly 865 870 875 gat gat cca ctt tct agg aag gac tct cgg tgg gga gat aga gat tca 2809 Asp Asp Pro Leu Ser Arg Lys Asp Ser Arg Trp Gly Asp Arg Asp Ser 880 885 890 gaa ggc acc tgg agg aaa gga cca gaa gct gac tct gag tgg aga aga 2857 Glu Gly Thr Trp Arg Lys Gly Pro Glu Ala Asp Ser Glu Trp Arg Arg 895 900 905 910 ggc cca cca gaa aag gag tgg aga cga gaa act cgg gat gat gag agg 2905 Gly Pro Pro Glu Lys Glu Trp Arg Arg Glu Thr Arg Asp Asp Glu Arg 915 920 925 cct cac agg aga gat gag gat cgg ctg agg cgc ttg ggg ggt gac gat 2953 Pro His Arg Arg Asp Glu Asp Arg Leu Arg Arg Leu Gly Gly Asp Asp 930 935 940 gaa gag aga gag tct tct ctt aga ccg gat gat gat cgt atc ccc agg 3001 Glu Glu Arg Glu Ser Ser Leu Arg Pro Asp Asp Asp Arg Ile Pro Arg 945 950 955 cgt ggc ctg gat gat gac aga ggg cct aga cgt ggt ccc gat gaa gat 3049 Arg Gly Leu Asp Asp Asp Arg Gly Pro Arg Arg Gly Pro Asp Glu Asp 960 965 970 aga ttt tct cgt aga gga aca gat gat gac cgt cct tcc tgg cgt aat 3097 Arg Phe Ser Arg Arg Gly Thr Asp Asp Asp Arg Pro Ser Trp Arg Asn 975 980 985 990 gca gat gat gat agg cct ccc agg cga att ggt gat gat gac agg gga 3145 Ala Asp Asp Asp Arg Pro Pro Arg Arg Ile Gly Asp Asp Asp Arg Gly 995 1000 1005 agc tgg cgt cac aca gat gac gac aga cca ccc aga cgg gga ctg gat 3193 Ser Trp Arg His Thr Asp Asp Asp Arg Pro Pro Arg Arg Gly Leu Asp 1010 1015 1020 gat gag aga gga agc tgg cgt aca gca gat gag gac aga gga cct aga 3241 Asp Glu Arg Gly Ser Trp Arg Thr Ala Asp Glu Asp Arg Gly Pro Arg 1025 1030 1035 cga ggg atg gat gat gac cgg ggg cca aga cga gga ggt gct gat gat 3289 Arg Gly Met Asp Asp Asp Arg Gly Pro Arg Arg Gly Gly Ala Asp Asp 1040 1045 1050 gaa cgg tca tct ggc gga atg ctg atg atg ata ggg gtc cca gga gag 3337 Glu Arg Ser Ser Gly Gly Met Leu Met Met Ile Gly Val Pro Gly Glu 1055 1060 1065 1070 gca tgg atg atg ata ggg gtc cca ggc gag ggc tgg atg atg acc gag 3385 Ala Trp Met Met Ile Gly Val Pro Gly Glu Gly Trp Met Met Thr Glu 1075 1080 1085 gac ctt gga gga atg ctg ctg aag ata gga ttt cca gga gag gtg cag 3433 Asp Leu Gly Gly Met Leu Leu Lys Ile Gly Phe Pro Gly Glu Val Gln 1090 1095 1100 atg atg acc gag ggc ctt gga gaa ata tgg atg atg ata ggg ttc cta 3481 Met Met Thr Glu Gly Leu Gly Glu Ile Trp Met Met Ile Gly Phe Leu 1105 1110 1115 gaa ggg gtg atg atg caa gac ctg gtc ctt gga gac cat ttg tca agc 3529 Glu Gly Val Met Met Gln Asp Leu Val Leu Gly Asp His Leu Ser Ser 1120 1125 1130 cag gtg gat gga gag aga aag aaa agg cta gag aag aga gtt ggg gtc 3577 Gln Val Asp Gly Glu Arg Lys Lys Arg Leu Glu Lys Arg Val Gly Val 1135 1140 1145 1150 cac ctc gag aat caa gac cat cag aag aac gtg aat ggg ata gag aca 3625 His Leu Glu Asn Gln Asp His Gln Lys Asn Val Asn Gly Ile Glu Thr 1155 1160 1165 aag aga agg aca gag ata atc aag atc gag agg aga atg aca aag acc 3673 Lys Arg Arg Thr Glu Ile Ile Lys Ile Glu Arg Arg Met Thr Lys Thr 1170 1175 1180 ttg aac gag ata ggg aca gag aga gag atg ggg acc ggg agg att cga 3721 Leu Asn Glu Ile Gly Thr Glu Arg Glu Met Gly Thr Gly Arg Ile Arg 1185 1190 1195 ttc aga aga ccc agg gat gaa ggt ggc tgg aga aga gga cca gca gaa 3769 Phe Arg Arg Pro Arg Asp Glu Gly Gly Trp Arg Arg Gly Pro Ala Glu 1200 1205 1210 gaa tct tca agc tgg aga gat tca agt cgc cgt gat gat agg gac agg 3817 Glu Ser Ser Ser Trp Arg Asp Ser Ser Arg Arg Asp Asp Arg Asp Arg 1215 1220 1225 1230 gaa gac cgt cga cgg gat aga gat gat cgt cgt gat ctg aga gac ctg 3865 Glu Asp Arg Arg Arg Asp Arg Asp Asp Arg Arg Asp Leu Arg Asp Leu 1235 1240 1245 aga gac aga agg gat tta aga gat gat aga gat cgg aga gga cct ccc 3913 Arg Asp Arg Arg Asp Leu Arg Asp Asp Arg Asp Arg Arg Gly Pro Pro 1250 1255 1260 ctc aga tca gag cga gaa gaa gca agc tct tgg aga cgc act gat gac 3961 Leu Arg Ser Glu Arg Glu Glu Ala Ser Ser Trp Arg Arg Thr Asp Asp 1265 1270 1275 agg aaa gat gac cgg act gaa gag agg gat cca cct cgt cgt gtt cct 4009 Arg Lys Asp Asp Arg Thr Glu Glu Arg Asp Pro Pro Arg Arg Val Pro 1280 1285 1290 ccc cca gct ctt tca aga gat cga gaa aga gag cga gaa cga gaa ggt 4057 Pro Pro Ala Leu Ser Arg Asp Arg Glu Arg Glu Arg Glu Arg Glu Gly 1295 1300 1305 1310 gag aaa gag aaa gca tcc tgg aga gct gag aaa gat agg gag tcc ctt 4105 Glu Lys Glu Lys Ala Ser Trp Arg Ala Glu Lys Asp Arg Glu Ser Leu 1315 1320 1325 cgt cgt act aag aat gaa act gat gaa gat gga tgg acc aca gta cga 4153 Arg Arg Thr Lys Asn Glu Thr Asp Glu Asp Gly Trp Thr Thr Val Arg 1330 1335 1340 cgt t aagtcccaag atgatggagt caaacttgtg gcttacatag gtttgatcac 4207 Arg attcaaggat tattatactt gtgcttcaac cagtctaaat tgaattcttt aatgttgtct 4267 caccataaca caaaaagcat gaacttgtat taatcatata taatagattg atcatgcact 4327 gtattcacag gaggttggaa aaccatgcca ttttctggaa cttaaggtgt tgcattattt 4387 catcaatcat ttgttaaaaa aaaaaaacta aaaaataaaa atgtgaaccc ttcagtgtaa 4447 acaccttatc ttggtataca atgatctttt tgttttgttt tgaagtatca gatattaatt 4507 tggaataagg taaggttctc ttgaaacatt tgaaaaccct ttaagccaac tgatctgaca 4567 gctttcccat cagtagaagt gggaacatac cttcttaggt atttactatt aactacatgt 4627 aggcagttta tagcttctga tcagttagta gacattacaa acactggttg taatggggtt 4687 ttctgtagac tttacttgag aggtgagtat aaagcatttt ttagtcatca tcatgacgat 4747 gctgctcaag tgcagatcca gaacagtaca gcgttgggtt cctagagcat ttggtaaact 4807 gttgtgggtt ttttctttct gttgccaaaa ctgcttttcc actaattcat gcctttcaag 4867 cattttaaat atgacaatat ttataaatgt gtggtttgga ggaattgttt aaattctttt 4927 tcctaatttt cttttttcag gatagattat ttcaacaagt aatttgtagt gatgactgtg 4987 ttgacttcaa ttttggagtg tagtagctgt gtttaaaaaa aagagtaata accatttggt 5047 cttattgaag ccaacacgga atttgctgct gtgttttttt ctttggtgat aaataaaata 5107 cttacataat tggaaaaaaa aaaaaaaaaa 5137 2 1343 PRT Mus sp. 2 Met Pro Ala Tyr Phe Gln Arg Pro Glu Asn Ala Leu Lys Arg Ala Asn 1 5 10 15 Glu Phe Leu Glu Val Gly Lys Lys Gln Pro Ala Leu Asp Val Leu Tyr 20 25 30 Asp Val Met Lys Ser Lys Lys His Arg Thr Trp Gln Lys Ile His Glu 35 40 45 Pro Ile Met Leu Lys Tyr Leu Glu Leu Cys Val Asp Leu Arg Lys Ser 50 55 60 His Leu Ala Lys Glu Gly Leu Tyr Gln Tyr Lys Asn Ile Cys Gln Gln 65 70 75 80 Val Asn Ile Lys Ser Leu Glu Asp Val Val Arg Ala Tyr Leu Lys Leu 85 90 95 Ala Glu Glu Lys Thr Glu Ala Ala Lys Glu Glu Ser Gln Gln Met Val 100 105 110 Leu Asp Ile Glu Asp Leu Asp Asn Ile Gln Thr Pro Glu Ser Val Leu 115 120 125 Leu Ser Ala Val Ser Gly Glu Asp Thr Gln Asp Arg Thr Asp Arg Leu 130 135 140 Leu Leu Thr Pro Trp Val Lys Phe Leu Trp Asp His Thr Gly Ser Val 145 150 155 160 Leu Asp Leu Leu Arg Asn Asn Ser Arg Val Glu Arg Leu Tyr His Asp 165 170 175 Ile Ala Gln Gln Ala Phe Lys Phe Cys Leu Gln Tyr Thr Arg Lys Ala 180 185 190 Glu Phe Arg Lys Leu Cys Asp Asn Leu Arg Ile Asp Leu Ser Gln Ile 195 200 205 Gln Arg His His Asn Gln Ser Thr Ala Ile Asn Leu Asn Asn Pro Glu 210 215 220 Ser Gln Ser Met His Leu Glu Thr Arg Leu Val Gln Leu Asp Ser Ala 225 230 235 240 Ile Ser Met Glu Leu Trp Gln Glu Ala Phe Lys Ala Val Glu Asp Ile 245 250 255 His Gly Leu Phe Ser Leu Ser Lys Lys Pro Pro Lys Pro Gln Leu Met 260 265 270 Ala Asn Tyr Tyr Asn Lys Val Ser Thr Val Phe Trp Lys Ser Gly Asn 275 280 285 Ala Leu Phe His Ala Ser Thr Leu His Arg Leu Tyr His Leu Ser Arg 290 295 300 Glu Met Arg Lys Asn Leu Thr Gln Glu Glu Met Gln Arg Met Ser Thr 305 310 315 320 Arg Val Leu Leu Ala Thr Leu Ser Ile Pro Ile Thr Pro Glu Arg Thr 325 330 335 Asp Ile Ala Arg Leu Leu Asp Met Asp Gly Ile Ile Val Glu Lys Gln 340 345 350 Arg Arg Leu Ala Thr Leu Leu Gly Leu Gln Ala Pro Pro Thr Arg Ile 355 360 365 Gly Leu Ile Asn Asp Met Val Arg Phe Ser Val Leu Gln Tyr Val Val 370 375 380 Pro Glu Val Lys Asp Leu Tyr Asn Trp Leu Glu Val Glu Phe Asn Pro 385 390 395 400 Leu Lys Leu Cys Glu Arg Val Thr Lys Val Leu Asn Trp Val Arg Glu 405 410 415 Gln Pro Glu Lys Glu Pro Glu Leu Gln Gln Tyr Val Pro Gln Leu Gln 420 425 430 Asn Asn Thr Ile Leu Arg Leu Leu Gln Gln Val Ala Gln Ile Tyr Gln 435 440 445 Ser Ile Glu Phe Ser Arg Leu Thr Ser Leu Val Pro Phe Val Asp Ala 450 455 460 Phe Gln Leu Glu Arg Ala Ile Val Asp Ala Ala Arg His Cys Asp Leu 465 470 475 480 Gln Val Arg Ile Asp His Thr Ser Arg Thr Leu Ser Phe Gly Ser Asp 485 490 495 Leu Asn Tyr Ala Thr Arg Glu Asp Ala Pro Val Gly Pro His Leu Gln 500 505 510 Ser Met Pro Ser Glu Gln Ile Arg Asn Gln Leu Thr Ala Met Ser Ser 515 520 525 Val Leu Ala Lys Ala Leu Glu Val Ile Arg Pro Ala His Ile Leu Gln 530 535 540 Glu Lys Glu Glu Gln His Gln Leu Ala Val Asn Ala Tyr Leu Lys Asn 545 550 555 560 Ser Arg Lys Glu His Gln Arg Ile Leu Ala Arg Arg Gln Thr Ile Glu 565 570 575 Glu Arg Lys Glu Arg Leu Glu Ser Leu Asn Ile Gln Arg Glu Lys Glu 580 585 590 Glu Leu Glu Gln Arg Glu Ala Glu Leu Gln Lys Tyr Glu Arg Leu Lys 595 600 605 Lys Lys Gly Cys Gln Glu Ala Lys Glu Arg Glu Lys Glu Arg Ile Leu 610 615 620 Gln Glu His Glu Gln Ile Lys Lys Lys Thr Val Arg Glu Arg Leu Glu 625 630 635 640 Gln Ile Lys Lys Thr Glu Leu Gly Ala Lys Ala Phe Lys Asp Ile Asp 645 650 655 Ile Glu Asp Leu Glu Glu Leu Asp Pro Asp Phe Ile Met Ala Lys Gln 660 665 670 Val Glu Gln Leu Glu Lys Glu Lys Lys Glu Leu Gln Glu Arg Leu Lys 675 680 685 Asn Gln Glu Lys Lys Ile Asp Tyr Phe Glu Arg Ala Lys Arg Leu Glu 690 695 700 Glu Ile Pro Leu Ile Lys Ser Ala Tyr Glu Glu Gln Arg Val Lys Asp 705 710 715 720 Met Asp Leu Trp Glu Gln Gln Glu Glu Glu Arg Ile Thr Thr Met Gln 725 730 735 Leu Glu Arg Glu Lys Ala Leu Glu His Lys Asn Arg Met Ser Arg Met 740 745 750 Leu Glu Asp Arg Asp Leu Phe Val Met Arg Leu Lys Ala Ala Arg Gln 755 760 765 Ser Val Tyr Glu Glu Lys Leu Lys Gln Phe Glu Glu Arg Leu Ala Glu 770 775 780 Glu Arg His Ser Arg Leu Glu Asp Arg Lys Arg Gln Arg Lys Glu Glu 785 790 795 800 Arg Lys Ile Thr Tyr Tyr Arg Glu Arg Lys Lys Lys Ser Arg Gly Gly 805 810 815 Gln Arg Ser Arg Cys Ser Lys Thr Glu Lys Lys Glu Asn Val Leu Arg 820 825 830 Glu Gln Asn Ala Glu Glu Glu Leu Arg Glu Tyr Gln Glu Arg Val Lys 835 840 845 Lys Leu Glu Glu Val Glu Arg Lys Lys Arg Gln Arg Glu Leu Glu Ile 850 855 860 Glu Glu Arg Glu Arg Arg Arg Glu Glu Glu Arg Arg Leu Gly Asp Asp 865 870 875 880 Pro Leu Ser Arg Lys Asp Ser Arg Trp Gly Asp Arg Asp Ser Glu Gly 885 890 895 Thr Trp Arg Lys Gly Pro Glu Ala Asp Ser Glu Trp Arg Arg Gly Pro 900 905 910 Pro Glu Lys Glu Trp Arg Arg Glu Thr Arg Asp Asp Glu Arg Pro His 915 920 925 Arg Arg Asp Glu Asp Arg Leu Arg Arg Leu Gly Gly Asp Asp Glu Glu 930 935 940 Arg Glu Ser Ser Leu Arg Pro Asp Asp Asp Arg Ile Pro Arg Arg Gly 945 950 955 960 Leu Asp Asp Asp Arg Gly Pro Arg Arg Gly Pro Asp Glu Asp Arg Phe 965 970 975 Ser Arg Arg Gly Thr Asp Asp Asp Arg Pro Ser Trp Arg Asn Ala Asp 980 985 990 Asp Asp Arg Pro Pro Arg Arg Ile Gly Asp Asp Asp Arg Gly Ser Trp 995 1000 1005 Arg His Thr Asp Asp Asp Arg Pro Pro Arg Arg Gly Leu Asp Asp Glu 1010 1015 1020 Arg Gly Ser Trp Arg Thr Ala Asp Glu Asp Arg Gly Pro Arg Arg Gly 1025 1030 1035 1040 Met Asp Asp Asp Arg Gly Pro Arg Arg Gly Gly Ala Asp Asp Glu Arg 1045 1050 1055 Ser Ser Gly Gly Met Leu Met Met Ile Gly Val Pro Gly Glu Ala Trp 1060 1065 1070 Met Met Ile Gly Val Pro Gly Glu Gly Trp Met Met Thr Glu Asp Leu 1075 1080 1085 Gly Gly Met Leu Leu Lys Ile Gly Phe Pro Gly Glu Val Gln Met Met 1090 1095 1100 Thr Glu Gly Leu Gly Glu Ile Trp Met Met Ile Gly Phe Leu Glu Gly 1105 1110 1115 1120 Val Met Met Gln Asp Leu Val Leu Gly Asp His Leu Ser Ser Gln Val 1125 1130 1135 Asp Gly Glu Arg Lys Lys Arg Leu Glu Lys Arg Val Gly Val His Leu 1140 1145 1150 Glu Asn Gln Asp His Gln Lys Asn Val Asn Gly Ile Glu Thr Lys Arg 1155 1160 1165 Arg Thr Glu Ile Ile Lys Ile Glu Arg Arg Met Thr Lys Thr Leu Asn 1170 1175 1180 Glu Ile Gly Thr Glu Arg Glu Met Gly Thr Gly Arg Ile Arg Phe Arg 1185 1190 1195 1200 Arg Pro Arg Asp Glu Gly Gly Trp Arg Arg Gly Pro Ala Glu Glu Ser 1205 1210 1215 Ser Ser Trp Arg Asp Ser Ser Arg Arg Asp Asp Arg Asp Arg Glu Asp 1220 1225 1230 Arg Arg Arg Asp Arg Asp Asp Arg Arg Asp Leu Arg Asp Leu Arg Asp 1235 1240 1245 Arg Arg Asp Leu Arg Asp Asp Arg Asp Arg Arg Gly Pro Pro Leu Arg 1250 1255 1260 Ser Glu Arg Glu Glu Ala Ser Ser Trp Arg Arg Thr Asp Asp Arg Lys 1265 1270 1275 1280 Asp Asp Arg Thr Glu Glu Arg Asp Pro Pro Arg Arg Val Pro Pro Pro 1285 1290 1295 Ala Leu Ser Arg Asp Arg Glu Arg Glu Arg Glu Arg Glu Gly Glu Lys 1300 1305 1310 Glu Lys Ala Ser Trp Arg Ala Glu Lys Asp Arg Glu Ser Leu Arg Arg 1315 1320 1325 Thr Lys Asn Glu Thr Asp Glu Asp Gly Trp Thr Thr Val Arg Arg 1330 1335 1340 3 276 PRT Mus sp. peptide 3 Met Pro Ser Glu Gln Ile Arg Asn Gln Leu Thr Ala Met Ser Ser Val 1 5 10 15 Leu Ala Lys Ala Ile Glu Val Ile Arg Pro Ala His Ile Leu Gln Glu 20 25 30 Lys Glu Glu Gln His Gln Leu Ala Val Asn Ala Tyr Leu Lys Asn Ser 35 40 45 Arg Lys Glu His Gln Arg Ile Leu Ala Arg Arg Gln Thr Ile Glu Glu 50 55 60 Arg Lys Glu Arg Leu Glu Ser Leu Asn Ile Gln Arg Glu Lys Glu Glu 65 70 75 80 Leu Glu Gln Arg Glu Ala Glu Leu Gln Lys Val Arg Lys Ala Glu Glu 85 90 95 Glu Arg Leu Pro Arg Gly Lys Gly Ala Arg Glu Gly Thr Asn Pro Ser 100 105 110 Arg Thr Arg Ala Asn Gln Glu Glu Asn Cys Ser Gly Ala Val Arg Ala 115 120 125 Asp Gln Glu Asp Arg Leu Gly Ala Lys Ala Phe Lys Asp Ile Asp Ile 130 135 140 Glu Asp Leu Glu Glu Leu Asp Pro Asp Phe Ile Met Ala Lys Gln Val 145 150 155 160 Glu Gln Leu Glu Lys Glu Lys Lys Asp Tyr Gln Glu Arg Leu Lys Asn 165 170 175 Gln Glu Lys Lys Ile Asp Tyr Phe Glu Arg Ala Lys Arg Leu Glu Glu 180 185 190 Ile Pro Leu Ile Lys Ser Ala Tyr Glu Glu His Arg Val Lys Asp Met 195 200 205 Asp Leu Trp Glu Gln Gln Glu Glu Glu Arg Ile Thr Thr Met Gln Leu 210 215 220 Glu Arg Glu Lys Ala Leu Glu His Lys Asn Arg Met Ser Arg Met Leu 225 230 235 240 Glu Asp Arg Asp Leu Phe Val Met Arg Leu Lys Val Ala Arg Gln Ser 245 250 255 Val Tyr Glu Glu Lys Leu Lys Gln Phe Glu Glu Arg Leu Ala Glu Glu 260 265 270 Ser Ile Val Ala 275 4 447 PRT Mus sp. peptide 4 Met Pro Ser Glu Gln Ile Arg Asn Gln Leu Thr Ala Met Ser Ser Val 1 5 10 15 Leu Ala Lys Ala Ile Glu Val Ile Arg Pro Ala His Ile Leu Gln Glu 20 25 30 Lys Glu Glu Gln His Gln Leu Ala Val Asn Ala Tyr Leu Lys Asn Ser 35 40 45 Arg Lys Glu His Gln Arg Ile Leu Ala Arg Arg Gln Thr Ile Glu Glu 50 55 60 Arg Lys Glu Arg Leu Glu Ser Leu Asn Ile Gln Arg Glu Lys Glu Glu 65 70 75 80 Leu Glu Gln Arg Glu Ala Glu Leu Gln Lys Val Arg Lys Ala Glu Glu 85 90 95 Glu Arg Leu Pro Arg Gly Lys Gly Ala Arg Glu Gly Thr Asn Pro Ser 100 105 110 Arg Thr Arg Ala Asn Gln Glu Glu Asn Cys Ser Gly Ala Val Arg Ala 115 120 125 Asp Gln Glu Asp Arg Leu Gly Ala Lys Ala Phe Lys Asp Ile Asp Ile 130 135 140 Glu Asp Leu Glu Glu Leu Asp Pro Asp Phe Ile Met Ala Lys Gln Val 145 150 155 160 Glu Gln Leu Glu Lys Glu Lys Lys Asp Tyr Gln Glu Arg Leu Lys Asn 165 170 175 Gln Glu Lys Lys Ile Asp Tyr Phe Glu Arg Ala Lys Arg Leu Glu Glu 180 185 190 Ile Pro Leu Ile Lys Ser Ala Tyr Glu Glu His Arg Val Lys Asp Met 195 200 205 Asp Leu Trp Glu Gln Gln Glu Glu Glu Arg Ile Thr Thr Met Gln Leu 210 215 220 Glu Arg Glu Lys Ala Leu Glu His Lys Asn Arg Met Ser Arg Met Leu 225 230 235 240 Glu Asp Arg Asp Leu Phe Val Met Arg Leu Lys Val Ala Arg Gln Ser 245 250 255 Val Tyr Glu Glu Lys Leu Lys Gln Phe Glu Glu Arg Leu Ala Glu Glu 260 265 270 Arg His Ser Arg Leu Glu Asp Arg Lys Arg Gln Arg Lys Glu Glu Arg 275 280 285 Lys Ile Thr Tyr Tyr Arg Glu Lys Glu Glu Glu Glu Gln Arg Arg Ala 290 295 300 Glu Glu Gln Met Leu Lys Glu Arg Glu Glu Arg Glu Arg Ala Glu Arg 305 310 315 320 Ala Lys Arg Glu Glu Glu Leu Arg Glu Tyr Gln Glu Arg Val Lys Lys 325 330 335 Leu Glu Glu Val Glu Arg Lys Lys Arg Gln Arg Glu Leu Glu Ile Glu 340 345 350 Glu Arg Glu Arg Arg Arg Glu Glu Glu Arg Arg Leu Gly Asp Asp Pro 355 360 365 Leu Ser Arg Lys Asp Ser Arg Trp Gly Asp Arg Asp Ser Glu Gly Thr 370 375 380 Trp Arg Lys Gly Pro Glu Ala Asp Ser Glu Trp Arg Arg Gly Pro Pro 385 390 395 400 Glu Lys Glu Trp Arg Arg Glu Thr Arg Asp Asp Glu Arg Pro His Arg 405 410 415 Arg Asp Glu Asp Arg Leu Arg Arg Leu Gly Gly Asp Asp Glu Glu Arg 420 425 430 Glu Ser Ser Leu Arg Pro Asp Asp Asp Arg Ile Pro Arg Arg Gly 435 440 445 5 964 PRT Saccharomyces sp. peptide 5 Met Ala Pro Pro Pro Phe Arg Pro Glu Asn Ala Ile Lys Arg Ala Asp 1 5 10 15 Glu Leu Ile Ser Val Gly Glu Lys Gln Ala Ala Leu Gln Ser Leu His 20 25 30 Asp Phe Ile Thr Ala Arg Arg Ile Arg Trp Ala Thr Pro Ser Thr Val 35 40 45 Glu Pro Val Val Phe Lys Phe Leu Glu Ile Gly Val Glu Leu Lys Lys 50 55 60 Gly Lys Leu Leu Lys Asp Gly Leu His Gln Tyr Lys Lys Leu Ile Gln 65 70 75 80 Gly Ser Thr Glu Gly Leu Val Ser Val Gly Ala Val Ala Arg Lys Phe 85 90 95 Ile Asp Leu Val Glu Ser Lys Ile Ala Ser Glu Gln Thr Arg Ala Asp 100 105 110 Glu Leu Gln Lys Gln Glu Ile Asp Asp Asp Leu Glu Gly Gly Val Thr 115 120 125 Pro Glu Asn Leu Leu Ile Ser Val Tyr Glu Ser Asp Gln Ser Val Ala 130 135 140 Gly Phe Asn Asp Glu Ala Ile Thr Ser Trp Leu Arg Phe Thr Trp Glu 145 150 155 160 Ser Tyr Arg Ala Val Leu Asp Leu Leu Arg Asn Asn Ala Leu Leu Glu 165 170 175 Ile Thr Tyr Ser Gly Val Val Lys Lys Thr Met His Phe Cys Leu Lys 180 185 190 Tyr Gln Arg Lys Asn Glu Phe Lys Arg Leu Ala Glu Met Leu Arg Gln 195 200 205 His Leu Asp Ala Ala Asn Tyr Gln Gln Ser Lys Ser Gly Asn Asn Leu 210 215 220 Val Asp Leu Ser Asp Ala Asp Thr Leu Gln Arg Tyr Leu Asp Gln Arg 225 230 235 240 Phe Gln Gln Val Asp Val Ser Val Lys Leu Glu Leu Trp His Glu Ala 245 250 255 Tyr Arg Ser Ile Glu Asp Val Phe His Leu Met Lys Ile Ser Lys Arg 260 265 270 Ala Pro Lys Pro Ser Thr Leu Ala Asn Tyr Tyr Glu Asn Leu Val Lys 275 280 285 Val Phe Phe Val Ser Gly Asp Pro Leu Leu His Thr Thr Ala Trp Lys 290 295 300 Lys Phe Tyr Lys Leu Tyr Ser Thr Asn Pro Arg Ala Thr Glu Glu Glu 305 310 315 320 Phe Lys Thr Tyr Ser Ser Thr Ile Phe Leu Ser Ala Ile Ser Thr Gln 325 330 335 Leu Asp Glu Ile Pro Ser Ile Gly Tyr Asp Pro His Leu Arg Met Tyr 340 345 350 Arg Leu Leu Asn Leu Asp Ala Lys Pro Thr Arg Lys Glu Met Leu Gln 355 360 365 Ser Ile Ile Glu Asp Glu Ser Ile Tyr Gly Lys Val Asp Glu Glu Leu 370 375 380 Lys Glu Leu Tyr Asp Ile Ile Glu Val Asn Phe Asp Val Asp Thr Val 385 390 395 400 Lys Gln Gln Leu Glu Asn Leu Leu Val Lys Leu Ser Ser Lys Thr Tyr 405 410 415 Phe Ser Gln Tyr Ile Ala Pro Leu Arg Asp Val Ile Met Arg Arg Val 420 425 430 Phe Val Ala Ala Ser Gln Lys Phe Thr Thr Val Ser Gln Ser Glu Leu 435 440 445 Tyr Lys Leu Ala Thr Leu Pro Ala Pro Leu Asp Leu Ser Ala Trp Asp 450 455 460 Ile Glu Lys Ser Leu Leu Gln Ala Ala Val Glu Asp Tyr Val Ser Ile 465 470 475 480 Thr Ile Asp His Glu Ser Ala Lys Val Thr Phe Ala Lys Asp Pro Phe 485 490 495 Asp Ile Phe Ala Ser Thr Ala Ser Lys Glu Val Ser Glu Glu Glu Asn 500 505 510 Thr Glu Pro Glu Val Gln Glu Glu Lys Glu Glu Thr Asp Glu Ala Leu 515 520 525 Gly Pro Gln Glu Thr Glu Asp Gly Glu Glu Lys Glu Glu Glu Ser Asp 530 535 540 Pro Val Ile Ile Arg Asn Ser Tyr Ile His Asn Lys Leu Leu Glu Leu 545 550 555 560 Ser Asn Val Leu His Asp Val Asp Ser Phe Asn Asn Ala Ser Tyr Met 565 570 575 Glu Lys Val Arg Ile Ala Arg Glu Thr Leu Ile Lys Lys Asn Lys Asp 580 585 590 Asp Leu Glu Lys Ile Ser Lys Ile Val Asp Glu Arg Val Lys Arg Ser 595 600 605 Gln Glu Gln Lys Gln Lys His Met Glu His Ala Ala Leu His Ala Glu 610 615 620 Gln Asp Ala Glu Val Arg Gln Gln Arg Ile Leu Glu Glu Lys Ala Ala 625 630 635 640 Ile Glu Ala Lys Leu Glu Glu Glu Ala His Arg Arg Leu Ile Glu Lys 645 650 655 Lys Lys Arg Glu Phe Glu Ala Ile Lys Glu Arg Glu Ile Thr Lys Met 660 665 670 Ile Thr Glu Val Asn Ala Lys Gly His Val Tyr Ile Asp Pro Asn Glu 675 680 685 Ala Lys Ser Leu Asp Leu Asp Thr Ile Lys Gln Val Ile Ile Ala Glu 690 695 700 Val Ser Lys Asn Lys Ser Glu Leu Glu Ser Arg Met Glu Tyr Ala Met 705 710 715 720 Lys Lys Leu Asp His Thr Glu Arg Ala Leu Arg Lys Val Glu Leu Pro 725 730 735 Leu Leu Gln Lys Glu Val Asp Lys Leu Gln Glu Thr Asp Thr Ala Asn 740 745 750 Tyr Glu Ala Met Lys Lys Lys Ile Val Asp Ala Ala Lys Ala Glu Tyr 755 760 765 Glu Ala Arg Met Ala Asp Arg Lys Asn Leu Val Met Val Tyr Asp Asp 770 775 780 Tyr Leu Lys Phe Lys Glu His Val Ser Gly Thr Lys Glu Ser Glu Leu 785 790 795 800 Ala Ala Ile Arg Asn Gln Lys Lys Ala Glu Leu Glu Ala Ala Lys Lys 805 810 815 Ala Arg Ile Glu Glu Val Arg Lys Arg Arg Tyr Glu Glu Ala Ile Ala 820 825 830 Arg Arg Lys Glu Glu Ile Ala Asn Ala Glu Arg Gln Lys Arg Ala Gln 835 840 845 Glu Leu Ala Glu Ala Thr Arg Lys Gln Arg Glu Ile Glu Glu Ala Ala 850 855 860 Ala Lys Lys Ser Thr Pro Tyr Ser Phe Arg Ala Gly Asn Arg Glu Pro 865 870 875 880 Pro Ser Thr Pro Ser Thr Leu Pro Lys Ala Thr Val Ser Pro Asp Lys 885 890 895 Ala Lys Leu Asp Met Ile Ala Gln Lys Gln Arg Glu Met Glu Glu Ala 900 905 910 Ile Glu Gln Arg Leu Ala Gly Arg Thr Ala Gly Gly Ser Ser Pro Ala 915 920 925 Thr Pro Ala Thr Pro Ala Thr Pro Ala Thr Pro Thr Pro Ser Ser Gly 930 935 940 Pro Lys Lys Met Thr Met Ala Glu Lys Leu Arg Ala Lys Arg Leu Ala 945 950 955 960 Lys Gly Gly Arg 6 958 PRT Nicotiana sp. peptide 6 Met Ala Thr Phe Ala Lys Pro Glu Asn Ala Leu Lys Arg Ala Glu Glu 1 5 10 15 Leu Ile Thr Val Gly Gln Lys Gln Glu Ala Leu Gln Ala Leu His Asp 20 25 30 Leu Ile Thr Ser Arg Arg Tyr Arg Ala Trp Gln Lys Thr Leu Glu Arg 35 40 45 Ile Met Phe Lys Tyr Val Glu Leu Cys Val Asp Met Arg Arg Gly Arg 50 55 60 Phe Ala Lys Asp Gly Leu Ile Gln Tyr Arg Ile Val Cys Gln Gln Val 65 70 75 80 Asn Ile Asn Ser Leu Glu Glu Val Ile Lys His Phe Met His Leu Ala 85 90 95 Thr Glu Arg Ala Glu Leu Ala Arg Asn Gln Ala Gln Ala Leu Glu Glu 100 105 110 Ala Leu Asp Val Glu Asp Leu Glu Ala Asp Lys Arg Pro Glu Asp Leu 115 120 125 Met Leu Ser Tyr Val Ser Gly Glu Lys Gly Lys Asp Arg Ser Asp Arg 130 135 140 Glu Leu Val Thr Pro Trp Phe Lys Phe Leu Trp Glu Thr Tyr Arg Thr 145 150 155 160 Val Leu Glu Ile Leu Arg Asn Asn Ser Arg Leu Glu Ala Leu Tyr Ala 165 170 175 Met Thr Ala His Arg Ala Phe Gln Phe Cys Lys Gln Tyr Lys Arg Thr 180 185 190 Thr Glu Phe Arg Arg Leu Cys Glu Ile Ile Arg Asn His Leu Ala Asn 195 200 205 Leu Asn Lys Tyr Arg Asp Gln Arg Asp Arg Pro Asp Leu Ser Ala Pro 210 215 220 Glu Ser Leu Gln Leu Tyr Leu Asp Thr Arg Phe Glu Gln Leu Lys Val 225 230 235 240 Ala Thr Glu Leu Gly Leu Trp Gln Glu Ala Phe Arg Ser Ile Glu Asp 245 250 255 Ile Tyr Gly Leu Met Cys Met Val Lys Lys Thr Pro Lys Ala Ser Leu 260 265 270 Met Val Val Tyr Tyr Gly Lys Leu Thr Glu Ile Phe Trp Met Ser Ser 275 280 285 Asn His Leu Tyr His Ala Tyr Ala Trp Leu Lys Leu Phe Ser Leu Gln 290 295 300 Lys Ser Phe Asn Lys Asn Leu Ser Gln Lys Asp Leu Gln Leu Ile Ala 305 310 315 320 Ser Ser Val Val Leu Ala Ala Leu Ser Val Pro Pro Tyr Asp Gln Ser 325 330 335 Tyr Gly Ala Ser His Leu Glu Leu Glu Asn Glu Lys Glu Arg Ser Leu 340 345 350 Arg Val Ala Asn Leu Ile Gly Phe Glu Val Glu Pro Lys Ala Glu Asn 355 360 365 Arg Val Ala Leu Ser Arg Ser Ser Leu Leu Ser Glu Leu Val Ser Lys 370 375 380 Gly Val Met Ser Cys Val Thr Gln Glu Val Lys Asp Leu Tyr His Leu 385 390 395 400 Leu Glu Asn Glu Phe Leu Pro Leu Asp Leu Ala Leu Lys Val Gln Pro 405 410 415 Val Leu Ser Lys Ile Ser Lys Leu Gly Gly Lys Leu Ser Ser Val Ser 420 425 430 Ser Val Pro Glu Val Gln Leu Ser Gln Tyr Val Pro Ala Leu Glu Lys 435 440 445 Leu Ala Thr Leu Arg Leu Leu Gln Gln Val Ser Gln Val Tyr Gln Thr 450 455 460 Ile Gln Ile Asp Asn Ile Ser Lys Met Ile Pro Phe Phe Asp Phe Thr 465 470 475 480 Val Ile Glu Lys Ile Ser Val Asp Ala Val Arg Arg Asn Phe Leu Ala 485 490 495 Ile Lys Val Asp His Met Lys Gly Leu Ser Ser Leu Val Asn Arg Val 500 505 510 Leu Arg Arg Lys Asp Ser Gly Ile Ile Cys Leu Phe Leu Ala Glu Ser 515 520 525 Leu Ser Lys Ala Arg Thr Met Ile Tyr Pro Pro Ala Lys Lys Ala Ala 530 535 540 Lys Leu Gly Glu Ala Leu Ser Asn Leu Ala Glu Ile Val Glu Lys Glu 545 550 555 560 His Lys Arg Leu Leu Ala Arg Lys Ser Ile Ile Glu Lys Arg Lys Glu 565 570 575 Glu Gln Glu Arg Leu Leu Leu Glu Met Glu Arg Val Glu Glu Thr Lys 580 585 590 Arg Arg Asp Val Gln Lys Met Thr Glu Glu Ala Glu Gln Lys Arg Ile 595 600 605 Ala Ala Glu Tyr Glu Gln Arg Arg Asn Gln Arg Ile Leu Lys Glu Ile 610 615 620 Glu Asp Arg Glu Leu Glu Glu Ala Gln Ala Leu Leu His Glu Ala Glu 625 630 635 640 Lys Arg Ser Lys Arg Lys Lys Lys Pro Val Leu Glu Gly Glu Lys Met 645 650 655 Thr Lys Lys Val Ile Met Glu Leu Ala Leu Asn Glu Gln Leu Arg Glu 660 665 670 Arg Gln Glu Met Glu Lys Lys Leu Leu Lys Phe Ala Lys Ser Met Asp 675 680 685 His Leu Glu Arg Ala Lys Arg Glu Glu Ala Ala Pro Leu Ile Glu Ser 690 695 700 Ala Phe Lys Gln Arg Leu Ala Glu Glu Ala Ala Leu His Glu Arg Glu 705 710 715 720 Gln Gln Gln Glu Ile Glu Leu Ser Arg Gln Arg His Ala Gly Asp Leu 725 730 735 Glu Glu Lys Arg Arg Leu Ala Arg Met Leu Glu Asn Lys Arg Ile Leu 740 745 750 Gln Glu Lys Val Val Ser Ser Arg Glu Ala Glu Phe Thr Arg Met Lys 755 760 765 Arg Glu Arg Gln Glu Arg Ile Ser Gln Ile Ile Gln Ser Arg Lys Gln 770 775 780 Glu Arg Glu Ala Arg Arg Lys Met Ile Phe Phe Leu Arg Ser Glu Glu 785 790 795 800 Glu Arg Gln Lys Arg Leu Gln Glu Glu Glu Glu Ala Arg Lys Arg Glu 805 810 815 Glu Ala Glu Arg Arg Lys Lys Glu Glu Ala Glu Arg Gln Ala Lys Leu 820 825 830 Asp Glu Ile Ala Glu Lys Gln Arg Arg Arg Met Leu Glu Leu Glu Glu 835 840 845 Lys Glu Lys Arg Glu Arg Glu Glu Ile Leu Arg Lys Ser Thr Ala Val 850 855 860 Leu Pro Lys Pro Ala Glu Pro Pro Thr Leu Gly Arg Pro Ala Glu Leu 865 870 875 880 Gly Gly Ala Ala Pro Ile Pro Ala Ala Ala Ala Thr Ala Pro Thr Pro 885 890 895 Gly Pro Gly Lys Tyr Val Pro Lys His Leu Arg Thr Lys Met Asp Gly 900 905 910 Ala Gly Gln Ala Pro Pro Pro Glu Thr Asp Lys Trp Gly Gly Gly Ser 915 920 925 Lys Pro Asp Asp Arg Pro Ser Trp Arg Asp Glu Arg Lys Pro Pro Ser 930 935 940 Phe Gly Ser Gly Ser Arg Thr Ser Trp Pro Ala Ser Arg Arg 945 950 955 7 1076 PRT Caenorhabditis elegans 7 Met Ala Pro Asn Tyr Phe Gln Lys Pro Glu Ala Ala Leu Lys Arg Ala 1 5 10 15 Glu Glu Leu Ile Gln Val Gly Lys Glu Ser Asp Ala Leu Asp Thr Leu 20 25 30 His Asp Thr Ile Lys Ala Arg Arg His Lys Gln Trp Thr Thr Val His 35 40 45 Glu Gln Ile Met Ile Lys His Met Glu Leu Cys Val Asp Leu Lys Lys 50 55 60 Gln His Leu Ala Lys Asp Ala Leu Phe Gln Tyr Lys Ala Leu Thr Gln 65 70 75 80 Gln Ile Asn Val Lys Ser Leu Glu Thr Val Val Val His Phe Leu Lys 85 90 95 Leu Ala Glu Gln Arg Thr Glu Asp Ala Gln Lys Gln Ser Ile Glu Lys 100 105 110 Val Glu Glu Ile Gly Asp Leu Asp Gln Gly Asp Val Pro Glu Arg Leu 115 120 125 Leu Leu Ala Val Val Ser Gly Ala Ala Ala Gln Asp Arg Met Asp Arg 130 135 140 Thr Val Leu Ala Pro Trp Leu Arg Phe Leu Trp Asp Ser Tyr Arg Asn 145 150 155 160 Cys Leu Glu Leu Leu Arg Asn Asn Ala Gln Val Glu Gln Leu Tyr His 165 170 175 Thr Ile Ser Arg His Ser Phe Thr Phe Cys Leu Arg Tyr Gln Arg Arg 180 185 190 Thr Glu Phe Arg Lys Leu Cys Asp Leu Leu Arg Met His Leu Asn Gln 195 200 205 Ile Gln Lys His Gln Tyr Ala Pro Asn Val Asn Ser Phe Arg Val Lys 210 215 220 Leu Thr Ser Pro Glu Ser Leu Gly Leu Met Gln Asp Thr Arg Leu Ile 225 230 235 240 Gln Leu Asp Thr Ala Ile Gln Met Glu Leu Trp Gln Glu Ala Tyr Lys 245 250 255 Ser Ala Glu Asp Val His Gly Met Met Gln Leu Ser Lys Asp Lys Asp 260 265 270 Lys Arg Thr Val Lys Pro Ala Ser Tyr Val Asn Tyr Tyr Asp Lys Leu 275 280 285 Ala Leu Val Phe Trp Lys Ala Gly Asn Ser Leu Phe His Ala Ala Ala 290 295 300 Leu Leu Gln Lys Phe Ile Ile Tyr Lys Asp Met Lys Lys Ser Phe Thr 305 310 315 320 Gln Asp Glu Ala Gln Glu Gln Ala Thr Arg Val Leu Leu Ala Thr Leu 325 330 335 Ser Ile Pro Glu Gly Ser Asp Ser Pro Ser Asp Leu Ser Arg Asn Leu 340 345 350 Asp Ile Glu Asp Gln His Val Ala Asn Met Arg Leu Leu Ser Asn Leu 355 360 365 Leu Arg Leu Pro Ile Ala Pro Thr Lys Asn Gly Ile Leu Lys Glu Ala 370 375 380 Ala Arg Ile Gly Val Pro Glu Ala Ala Gly Gln Thr Ala Lys Asp Leu 385 390 395 400 Tyr Lys Leu Leu Glu Ser Asn Phe Ser Pro Leu Lys Val Ala Lys Asp 405 410 415 Val Gln Ser Val Leu Asp Thr Val Thr Arg Pro Asp His Leu Gln Tyr 420 425 430 Val Glu Ser Leu Gln Ala Val Ala Ala Val Lys Ala Leu Lys Gln Val 435 440 445 Ser Val Ile Tyr Glu Ala Ile Ser Trp Glu Arg Ile Arg Lys Ile Ile 450 455 460 Pro Phe Tyr Ser Asp Leu Ala Leu Glu Arg Leu Val Val Glu Ala Ser 465 470 475 480 Lys His Arg Ile Val Lys Ala Gln Leu Asp His Arg Ala Asp Cys Val 485 490 495 Arg Phe Gly Ser Ser Asp Ala Thr Leu Ala Gly Gly Val Asp Glu Cys 500 505 510 Asp Asn Asn Glu Gly Phe Thr Gly Asp Asp Thr Gln Leu Gly Val Glu 515 520 525 Gly Val Arg Asn His Leu Glu Ala Met Tyr Thr Arg Leu Arg Gly Leu 530 535 540 Val Glu Gly Leu Asp Ala Glu Lys Arg Arg Lys Glu Ile Leu Lys Lys 545 550 555 560 Ile Glu Gly Gln Val Thr Ser Tyr Glu Lys Asn Arg Pro Thr Glu Ile 565 570 575 Glu Arg Ile His Arg Arg Lys Lys Met Leu Glu Asn Tyr Lys Glu Asn 580 585 590 Trp Glu Arg Val Lys Ala Glu Lys Thr Ala Ala Ala Ala Thr Glu Gln 595 600 605 Ala Lys Arg Glu Glu Ala Ala Arg Ala Glu Glu Met Lys Arg Leu Asp 610 615 620 Glu Gln Asn Lys Glu Ser Glu Arg Lys Arg Lys Gln Ala Glu Gln Asp 625 630 635 640 Glu Ile Gln Lys Lys Ile Lys Gln Asp Gln Leu Tyr Lys Met Gln Gln 645 650 655 Asn Ala Ile Tyr Gln Glu Ile Ile Lys Glu Lys Gly Leu Glu Gln Phe 660 665 670 Arg Asp Met Asp Pro Glu Gln Val Leu Arg Glu Gln Arg Glu Arg Leu 675 680 685 Asp Lys Glu Arg Ala Glu Thr Gln Arg Arg Leu Gln Gln Gln Glu Lys 690 695 700 Asn Phe Asp His His Val Arg Ala Leu His Leu Glu Glu Leu Asn Glu 705 710 715 720 Arg Arg Ala Val Met Asn Met Arg Leu Ser Glu Ala Pro Lys Leu His 725 730 735 Asp Leu Tyr Glu Glu Ala Arg Ile Ala Lys Glu Ile Ala Ala His Asp 740 745 750 Ser His Val Lys Leu Trp Gly Met Trp Asp Gln Val Arg Asp Ala Thr 755 760 765 Phe Asp Trp Val Glu Ser Val Lys Ile Asp Asn Gln Glu Thr Leu Glu 770 775 780 Lys Lys Leu Ser Asp Trp Gln Ala Lys Leu Glu Ala Val Arg Asn Asn 785 790 795 800 Arg Leu Ala Glu Arg Ala Glu Asp Arg Lys Lys Lys Arg Lys Glu Asp 805 810 815 Ala Ile Gln Ala Lys Ile Ala Glu Glu Arg Lys Lys Arg Glu Glu Glu 820 825 830 Glu Arg Ala Arg Leu Gln Val Ile Glu Gly Gln Arg Arg Gln His Asn 835 840 845 Asp Gly Arg Gly Arg Arg Glu Met Glu Asn Ser Val Ala Met Gln Asp 850 855 860 Asn Asp Trp Arg Arg Asn Pro Pro Arg Glu Ser Leu Pro Pro Arg Glu 865 870 875 880 Thr Arg Pro Met Arg Asp Gly Pro Thr Arg Glu Pro Arg Glu Phe Arg 885 890 895 Gly Asp Arg Asp Arg Glu Pro Arg Glu Pro Phe Arg Glu Val Pro Ser 900 905 910 Ser Lys Ala Asp Thr Asp Asn Ser Trp Arg Ser Ser Ala Gln Pro Thr 915 920 925 Arg Lys Pro Asp Asp Arg Arg Ser Asp Glu Phe Arg Arg Asn Asp Asp 930 935 940 Val Arg Arg Asn Asp Asp Val Arg Arg Asn Asp Pro Pro Arg Pro Ala 945 950 955 960 Ser Lys Ala Asp Thr Gly Asp Lys Trp Glu Arg Gly Val Lys Pro Val 965 970 975 Val Ser Pro Pro Lys Thr Asp Ala Pro Ser Val Ser Glu Pro Lys Ser 980 985 990 Glu Gly Pro Lys Arg Phe Val Pro Pro His Leu Arg Asn Arg Gln Gly 995 1000 1005 Gly Gly Gly Ala Gly Gly Ser Glu Glu Gln Ser Ala Pro Ala Arg Ser 1010 1015 1020 Gly Asn Ser Asn Val Thr Ser Pro Pro Asp Arg Ala Gln Gly Leu Arg 1025 1030 1035 1040 Gly Pro Pro Pro Thr Gly Arg Asn Ser Leu Pro Arg Arg Asp Gly Pro 1045 1050 1055 Pro Pro Arg Ser Asn Asn Thr Gly Asn Thr Gly Asn Ala Asp Ser Gly 1060 1065 1070 Ser Trp Arg Lys 1075 8 5301 DNA Homo sapiens mat_peptide (127)..(4272) 8 ccggctgggc gcgggcatct gctggcgagg cgcgtgggac cttacgctgg ttccccttcg 60 tctcctctcc cggcccgggc cactagagag ttcgctgacg ccgggtgagc tgagcctgcc 120 gccaagatgc cggcctattt ccagaggccg gaaaatgccc tcaaacgcgc caacgaattt 180 cttgaggttg gcaaaaagca gcctgctctg gatgttcttt atgatgttat gaaaagtaaa 240 aaacatagaa catggcaaaa gatacacgaa ccaattatgt tgaaatactt ggaactttgc 300 gtggatcttc gcaagagcca cttggcaaag gaggggttat accagtataa gaacatttgt 360 caacaggtga acataaaatc tctggaggat gttgttaggg catatttgaa aatggcagag 420 gaaaaaactg aagctgctaa agaagaatct cagcagatgg tcttagatat agaggatcta 480 gataatattc aaactcctga gagtgttctc ctaagtgctg taagtggtga agacactcag 540 gatcgtactg acagattact tttaactcca tgggttaaat tcctgtggga gtcttacagg 600 cagtgtttgg accttcttag aaacaattct agagtagagc gcctgtacca tgatattgcc 660 cagcaagctt tcaaattctg cctccaatac acgcgtaagg ctgaattccg taaactgtgt 720 gacaatttga gaatgcactt atcgcagatt cagcgccacc ataaccaaag tacggcaatc 780 aatcttaata atccagagag ccagtccatg catttggaaa ccagacttgt tcagctggac 840 agtgctatca gcatggaatt gtggcaggaa gcattcaaag ctgtggaaga tattcacggg 900 ctattctcct tgtctaaaaa accacctaaa cctcagttga tggcaaatta ctataacaaa 960 gtctcaactg tgttttggaa atctggaaat gctctttttc atgcatctac actccatcgt 1020 ctttaccatc tctctagaga aatgagaaag aatctcacac aagacgagat gcaaagaatg 1080 tctactagag tccttttagc cactctttcc atccctatta ctcctgagcg tacggatatt 1140 gctcgacttc tggatacgga tggcattata gttgaaaaac agcgtcgcct tgcaacacta 1200 ctaggtcttc aagccccacc gacacgaatt ggccttatta atgatatggt cagatttaat 1260 gtactacaat atgttgtccc agaagtgaaa gacctttaca attggcttga agtagaattt 1320 aacccattaa aactctgtga gcgagtcaca aaggttctaa attgggttag ggaacaacct 1380 gaaaaggaac cggaattgca gcagtatgtg ccacaactgc aaaacaacac catcctccac 1440 cttctgcagc aggtgtcaca gatttatcag agcattgagt tttctcgttt gacttctttg 1500 gttccttttg ttgatgcttt ccaactggaa cgggccatag tagatgcagc caggcattgc 1560 gacttgcagg ttcgtattga tcacacttct cggaccctga gttttggatc tgatttgaat 1620 tatgctactc gagaagatgc tccgattggt cctcatttgc aaagcatgcc ttcagagcag 1680 ataagaaacc agctgacagc catgtcctca gtacttgcaa aagcacttga agtcattaaa 1740 ccagctcata tactgcaaga gaaagaagaa cagcatcagt tggctgccac tgcatacctt 1800 aaaaattcac gaaaagagca ccagcggatc ctggctcgcc gccagacaat tgaggagaga 1860 aaagagcgcc ttgagagtct gaatattcag cgtgagaaag aagaattgga acagagggaa 1920 gctgaactcc agaaagtgcg gaaggctgag gaagagaggc tgcgccagga agcaaaggag 1980 agagagaagg agcgtatctt acaggaacat gaacaaatca aaaagaaaac tgtccgagag 2040 cgtttggagc agatcaagaa aacagaactg ggtgccaaag cattcaaaga tattgatatt 2100 gaagaccttg aggaattgga tccagatttt atcatggcta aacaggttga acaactggag 2160 aaagaaaaga aagaacttca agaacgccta aagaatcaag aaaagaagat tgactatttt 2220 gaaagagcca aacgtttgga agaaattcct ttgataaaga gcgcttacga ggaacagaga 2280 attaaagaca tggatctgtg ggagcaacaa gaggaagaaa gaattactac aatgcagcta 2340 gaacgtgaaa aggctcttga acataagaat cgaatgtcac gaatgcttga agacagagat 2400 ttattcgtaa tgcgactcaa agctgcacgg cagtctgttt atgaggaaaa acttaaacag 2460 tttgaagagc gattagcaga agaaaggcat aatcgattgg aagaacggaa aaggcagcgt 2520 aaagaagaac gcaggataac atactataga gaaaaagaag aggaggagca gagaagggca 2580 ggagaacaaa tgctacaaga gcgggaagag agagagcgcg ccgaacgagc aaaacgcgag 2640 gaagagctac gagagtatca ggagcgggtg aagaaattag aagaagtgga aaggaaaaaa 2700 cgccaaaggg agttggaaat tgaagaacga gaacggcgta gagaggaaga gagaagactt 2760 ggcgatagtt ccctttctag aaaggactct cgttggggag atagagattc agaaggcacc 2820 tggagaaaag gacctgaagc agattctgag tggagaagag gcccgccaga gaaggagtgg 2880 agacgtggag aagggcgaga tgaggacagg tctcatagaa gagatgaaga gcggccccgg 2940 cgtctggggg atgttgaaga tagagagccc tctcttagac cagacgatga tcgggttccc 3000 cggcgtggca tggatgatga cagaggccct agacgtggtc ctgaggaaga taggttctct 3060 cgtcgtgggg cagacgatga ccggccttcc tggcgtaaca cagatgatga caggcctccc 3120 agacgaattg ccgatgaaga caggggaaac tggcgtcatg cggatgatga cagaccacct 3180 agacgaggac tggatgagga cagaggaagc tggcgaacag ctgatgagga cagaggacca 3240 agacgtggga tggatgatga ccgggggccg aggcgaggag gcgctgatga tgagcgatca 3300 tcctggcgta atgctgatga tgaccggggt cccaggcgag ggttggatga tgatcggggt 3360 cccaggcgag gcatggatga tgaccggggt cccaggcgag gcatggatga tgaccggggt 3420 cccaggcgag gcatggatga tgaccggggt cccaggcgag ggttggatga tgatcgagga 3480 ccttggagga acgccgatga tgacagaatt cccaggcgtg gtgcagagga tgacaggggc 3540 ccttggagaa acatggatga tgatcgcctt tcaagacgtg ctgatgatga tcggtttccc 3600 agacggggtg atgactcaag acctggtcct tggagaccat tagtcaagcc aggtggatgg 3660 agagagaaag aaaaagccag agaggagagc tggggtccac ctcgagaatc aaggccatca 3720 gaagaacgtg aatgggacag agaaaaagaa agggacagag ataatcaaga tcgggaggag 3780 aatgacaagg accctgagag agaaagggac agagagagag atgtggatcg agaggatcgc 3840 ttcagaagac ctagggatga aggtggctgg agaagaggac cagctgagga atcttcaagc 3900 tggagagact caagtcgccg ggacgatagg gatagggatg accgtcgccg tgagagggat 3960 gaccggcgtg atctaagaga aagacgagat ctaagagacg acagggaccg aagaggacct 4020 ccactcagat cagaacgtga agaagtaagt tcttggagac gtgctgatga caggaaagat 4080 gaccgggtgg aagagcggga ccctcctcgt cgagttcctc ccccagctct ttcaagagac 4140 cgagaaagag accgagaccg agaaagagaa ggtgaaaaag agaaggcctc atggagagct 4200 gagaaagata gggaatctct ccgtcgtact aaaaatgaga ctgatgaaga tggatggacc 4260 acagtacgac gttaacccgg gcgcgtctca agataatgga tttaaactgg tgtcttaaat 4320 aggtttgatc acattcaagg attattatac ttgtgcttca accaatctaa attggattct 4380 ttaatgttgt ttcaccataa cacaaaaagc atgaacttgt attaatccta tataatagat 4440 tgatcatgca ccatatccac aggaggttgg aaaaaccatg ccattttctg gaatttaagg 4500 gtgttgcatt atttcatcaa tcatttgttg acaaaaaaga aaaactaaaa aataaattta 4560 aaatgtgaac cttcaggtat tgagtaacac ctttatcttg gtatagaact gatacttttt 4620 tttgattttg aaatatctga taataatttg gaatgaagta aggtcctgtt aaaatatatt 4680 tgaagaccct ttaaagcagt gaatctgaaa caattttcac acccttaagt ggttgatacg 4740 tacctatttt aggtattttg aggtatttac cataaactaa atttagaaat tttttagatt 4800 cacttgaagt aaacattaca aacattggat acggtggggt tttctttaga ttttacttga 4860 gagaaggtga gtacaaagca atttgcagtt gttgtaatga caagattact gcgcaagtgt 4920 gaatccaaac agtatagctt ttaaatttta aagcatttgg taaattatcg ctgagttttt 4980 ttctgttgcc aatagcaaac tgcttttcca ttaatggaga attcatgcct ttcaagcatt 5040 ttaaatatga caatatttat aaatgtatgg tttggaggaa tcgtttaaat tctctttcct 5100 aattttcttt cttttgaaga tagattcttt caacaagtaa tttgtagtaa tgactgtgtt 5160 gacttcaatt ttggagcgca gtagctatgt taaagatgaa ctatttggtc tcattgaagc 5220 caacacagaa cttgctgctg tgttttttct tcagtgataa ataaaatact taccaaaaaa 5280 aaaaaaaaaa aaaaaaaaaa a 5301 9 1382 PRT Homo sapiens 9 Met Pro Ala Tyr Phe Gln Arg Pro Glu Asn Ala Leu Lys Arg Ala Asn 1 5 10 15 Glu Phe Leu Glu Val Gly Lys Lys Gln Pro Ala Leu Asp Val Leu Tyr 20 25 30 Asp Val Met Lys Ser Lys Lys His Arg Thr Trp Gln Lys Ile His Glu 35 40 45 Pro Ile Met Leu Lys Tyr Leu Glu Leu Cys Val Asp Leu Arg Lys Ser 50 55 60 His Leu Ala Lys Glu Gly Leu Tyr Gln Tyr Lys Asn Ile Cys Gln Gln 65 70 75 80 Val Asn Ile Lys Ser Leu Glu Asp Val Val Arg Ala Tyr Leu Lys Met 85 90 95 Ala Glu Glu Lys Thr Glu Ala Ala Lys Glu Glu Ser Gln Gln Met Val 100 105 110 Leu Asp Ile Glu Asp Leu Asp Asn Ile Gln Thr Pro Glu Ser Val Leu 115 120 125 Leu Ser Ala Val Ser Gly Glu Asp Thr Gln Asp Arg Thr Asp Arg Leu 130 135 140 Leu Leu Thr Pro Trp Val Lys Phe Leu Trp Glu Ser Tyr Arg Gln Cys 145 150 155 160 Leu Asp Leu Leu Arg Asn Asn Ser Arg Val Glu Arg Leu Tyr His Asp 165 170 175 Ile Ala Gln Gln Ala Phe Lys Phe Cys Leu Gln Tyr Thr Arg Lys Ala 180 185 190 Glu Phe Arg Lys Leu Cys Asp Asn Leu Arg Met His Leu Ser Gln Ile 195 200 205 Gln Arg His His Asn Gln Ser Thr Ala Ile Asn Leu Asn Asn Pro Glu 210 215 220 Ser Gln Ser Met His Leu Glu Thr Arg Leu Val Gln Leu Asp Ser Ala 225 230 235 240 Ile Ser Met Glu Leu Trp Gln Glu Ala Phe Lys Ala Val Glu Asp Ile 245 250 255 His Gly Leu Phe Ser Leu Ser Lys Lys Pro Pro Lys Pro Gln Leu Met 260 265 270 Ala Asn Tyr Tyr Asn Lys Val Ser Thr Val Phe Trp Lys Ser Gly Asn 275 280 285 Ala Leu Phe His Ala Ser Thr Leu His Arg Leu Tyr His Leu Ser Arg 290 295 300 Glu Met Arg Lys Asn Leu Thr Gln Asp Glu Met Gln Arg Met Ser Thr 305 310 315 320 Arg Val Leu Leu Ala Thr Leu Ser Ile Pro Ile Thr Pro Glu Arg Thr 325 330 335 Asp Ile Ala Arg Leu Leu Asp Thr Asp Gly Ile Ile Val Glu Lys Gln 340 345 350 Arg Arg Leu Ala Thr Leu Leu Gly Leu Gln Ala Pro Pro Thr Arg Ile 355 360 365 Gly Leu Ile Asn Asp Met Val Arg Phe Asn Val Leu Gln Tyr Val Val 370 375 380 Pro Glu Val Lys Asp Leu Tyr Asn Trp Leu Glu Val Glu Phe Asn Pro 385 390 395 400 Leu Lys Leu Cys Glu Arg Val Thr Lys Val Leu Asn Trp Val Arg Glu 405 410 415 Gln Pro Glu Lys Glu Pro Glu Leu Gln Gln Tyr Val Pro Gln Leu Gln 420 425 430 Asn Asn Thr Ile Leu His Leu Leu Gln Gln Val Ser Gln Ile Tyr Gln 435 440 445 Ser Ile Glu Phe Ser Arg Leu Thr Ser Leu Val Pro Phe Val Asp Ala 450 455 460 Phe Gln Leu Glu Arg Ala Ile Val Asp Ala Ala Arg His Cys Asp Leu 465 470 475 480 Gln Val Arg Ile Asp His Thr Ser Arg Thr Leu Ser Phe Gly Ser Asp 485 490 495 Leu Asn Tyr Ala Thr Arg Glu Asp Ala Pro Ile Gly Pro His Leu Gln 500 505 510 Ser Met Pro Ser Glu Gln Ile Arg Asn Gln Leu Thr Ala Met Ser Ser 515 520 525 Val Leu Ala Lys Ala Leu Glu Val Ile Lys Pro Ala His Ile Leu Gln 530 535 540 Glu Lys Glu Glu Gln His Gln Leu Ala Ala Thr Ala Tyr Leu Lys Asn 545 550 555 560 Ser Arg Lys Glu His Gln Arg Ile Leu Ala Arg Arg Gln Thr Ile Glu 565 570 575 Glu Arg Lys Glu Arg Leu Glu Ser Leu Asn Ile Gln Arg Glu Lys Glu 580 585 590 Glu Leu Glu Gln Arg Glu Ala Glu Leu Gln Lys Val Arg Lys Ala Glu 595 600 605 Glu Glu Arg Leu Arg Gln Glu Ala Lys Glu Arg Glu Lys Glu Arg Ile 610 615 620 Leu Gln Glu His Glu Gln Ile Lys Lys Lys Thr Val Arg Glu Arg Leu 625 630 635 640 Glu Gln Ile Lys Lys Thr Glu Leu Gly Ala Lys Ala Phe Lys Asp Ile 645 650 655 Asp Ile Glu Asp Leu Glu Glu Leu Asp Pro Asp Phe Ile Met Ala Lys 660 665 670 Gln Val Glu Gln Leu Glu Lys Glu Lys Lys Glu Leu Gln Glu Arg Leu 675 680 685 Lys Asn Gln Glu Lys Lys Ile Asp Tyr Phe Glu Arg Ala Lys Arg Leu 690 695 700 Glu Glu Ile Pro Leu Ile Lys Ser Ala Tyr Glu Glu Gln Arg Ile Lys 705 710 715 720 Asp Met Asp Leu Trp Glu Gln Gln Glu Glu Glu Arg Ile Thr Thr Met 725 730 735 Gln Leu Glu Arg Glu Lys Ala Leu Glu His Lys Asn Arg Met Ser Arg 740 745 750 Met Leu Glu Asp Arg Asp Leu Phe Val Met Arg Leu Lys Ala Ala Arg 755 760 765 Gln Ser Val Tyr Glu Glu Lys Leu Lys Gln Phe Glu Glu Arg Leu Ala 770 775 780 Glu Glu Arg His Asn Arg Leu Glu Glu Arg Lys Arg Gln Arg Lys Glu 785 790 795 800 Glu Arg Arg Ile Thr Tyr Tyr Arg Glu Lys Glu Glu Glu Glu Gln Arg 805 810 815 Arg Ala Gly Glu Gln Met Leu Gln Glu Arg Glu Glu Arg Glu Arg Ala 820 825 830 Glu Arg Ala Lys Arg Glu Glu Glu Leu Arg Glu Tyr Gln Glu Arg Val 835 840 845 Lys Lys Leu Glu Glu Val Glu Arg Lys Lys Arg Gln Arg Glu Leu Glu 850 855 860 Ile Glu Glu Arg Glu Arg Arg Arg Glu Glu Glu Arg Arg Leu Gly Asp 865 870 875 880 Ser Ser Leu Ser Arg Lys Asp Ser Arg Trp Gly Asp Arg Asp Ser Glu 885 890 895 Gly Thr Trp Arg Lys Gly Pro Glu Ala Asp Ser Glu Trp Arg Arg Gly 900 905 910 Pro Pro Glu Lys Glu Trp Arg Arg Gly Glu Gly Arg Asp Glu Asp Arg 915 920 925 Ser His Arg Arg Asp Glu Glu Arg Pro Arg Arg Leu Gly Asp Val Glu 930 935 940 Asp Arg Glu Pro Ser Leu Arg Pro Asp Asp Asp Arg Val Pro Arg Arg 945 950 955 960 Gly Met Asp Asp Asp Arg Gly Pro Arg Arg Gly Pro Glu Glu Asp Arg 965 970 975 Phe Ser Arg Arg Gly Ala Asp Asp Asp Arg Pro Ser Trp Arg Asn Thr 980 985 990 Asp Asp Asp Arg Pro Pro Arg Arg Ile Ala Asp Glu Asp Arg Gly Asn 995 1000 1005 Trp Arg His Ala Asp Asp Asp Arg Pro Pro Arg Arg Gly Leu Asp Glu 1010 1015 1020 Asp Arg Gly Ser Trp Arg Thr Ala Asp Glu Asp Arg Gly Pro Arg Arg 1025 1030 1035 1040 Gly Met Asp Asp Asp Arg Gly Pro Arg Arg Gly Gly Ala Asp Asp Glu 1045 1050 1055 Arg Ser Ser Trp Arg Asn Ala Asp Asp Asp Arg Gly Pro Arg Arg Gly 1060 1065 1070 Leu Asp Asp Asp Arg Gly Pro Arg Arg Gly Met Asp Asp Asp Arg Gly 1075 1080 1085 Pro Arg Arg Gly Met Asp Asp Asp Arg Gly Pro Arg Arg Gly Met Asp 1090 1095 1100 Asp Asp Arg Gly Pro Arg Arg Gly Leu Asp Asp Asp Arg Gly Pro Trp 1105 1110 1115 1120 Arg Asn Ala Asp Asp Asp Arg Ile Pro Arg Arg Gly Ala Glu Asp Asp 1125 1130 1135 Arg Gly Pro Trp Arg Asn Met Asp Asp Asp Arg Leu Ser Arg Arg Ala 1140 1145 1150 Asp Asp Asp Arg Phe Pro Arg Arg Gly Asp Asp Ser Arg Pro Gly Pro 1155 1160 1165 Trp Arg Pro Leu Val Lys Pro Gly Gly Trp Arg Glu Lys Glu Lys Ala 1170 1175 1180 Arg Glu Glu Ser Trp Gly Pro Pro Arg Glu Ser Arg Pro Ser Glu Glu 1185 1190 1195 1200 Arg Glu Trp Asp Arg Glu Lys Glu Arg Asp Arg Asp Asn Gln Asp Arg 1205 1210 1215 Glu Glu Asn Asp Lys Asp Pro Glu Arg Glu Arg Asp Arg Glu Arg Asp 1220 1225 1230 Val Asp Arg Glu Asp Arg Phe Arg Arg Pro Arg Asp Glu Gly Gly Trp 1235 1240 1245 Arg Arg Gly Pro Ala Glu Glu Ser Ser Ser Trp Arg Asp Ser Ser Arg 1250 1255 1260 Arg Asp Asp Arg Asp Arg Asp Asp Arg Arg Arg Glu Arg Asp Asp Arg 1265 1270 1275 1280 Arg Asp Leu Arg Glu Arg Arg Asp Leu Arg Asp Asp Arg Asp Arg Arg 1285 1290 1295 Gly Pro Pro Leu Arg Ser Glu Arg Glu Glu Val Ser Ser Trp Arg Arg 1300 1305 1310 Ala Asp Asp Arg Lys Asp Asp Arg Val Glu Glu Arg Asp Pro Pro Arg 1315 1320 1325 Arg Val Pro Pro Pro Ala Leu Ser Arg Asp Arg Glu Arg Asp Arg Asp 1330 1335 1340 Arg Glu Arg Glu Gly Glu Lys Glu Lys Ala Ser Trp Arg Ala Glu Lys 1345 1350 1355 1360 Asp Arg Glu Ser Leu Arg Arg Thr Lys Asn Glu Thr Asp Glu Asp Gly 1365 1370 1375 Trp Thr Thr Val Arg Arg 1380 

What is claimed is:
 1. A method of detecting cells of metastatic potential comprising the steps of: (a) contacting an antibody of accession no. DSM2256 or DSM2257 with a cell or tissue sample, said antibody being capable of specifically recognizing an epitope of a polypeptide comprising an amino acid sequence encoded by a nucleotide sequence according to SEQ ID No:1 or SEQ ID No:8, and (b) detecting binding of said antibody to said cell or tissue sample.
 2. An isolated monoclonal antibody obtained from a hybridoma deposited under accession nos. DSM2256 or DSM2257.
 3. A method of identifying cells with metastatic potential comprising the steps of: (i) providing an antibody of accession nos. DSM2256 or DSM2257 capable of specifically recognizing an epitope of a cell-membrane associated polypeptide encoded by a nucleic acid sequence according to SEQ ID No: 1 or SEQ ID No: 8, (ii) incubating said antibody with cell of interest, and (iii) detecting binding of said antibody to said cells, wherein the antibody binding indicates said cells have metastatic potenial. 